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Direct Observation of Lipid Bilayer Disruption by Poly(amidoamine) Dendrimers
Abstract
Atomic force microscopy (AFM) is employed to observe the effect of poly(amidoamine) (PAMAM) dendrimers on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers. Aqueous solutions of generation 7 PAMAM dendrimers cause the formation of holes 15-40 nm in diameter in previously intact bilayers. This effect is observed for two different branch end-groups--amine and carboxyl. In contrast, carboxyl-terminated core-shell tectodendrimer clusters do not create holes in the lipid membrane but instead show a strong affinity to adsorb to the edges of existing bilayer defects. A possible mechanism for the formation of holes in the lipid bilayer is proposed. The dendrimers remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules. Dynamic light scattering (DLS) measurements as well as 31P NMR data support this explanation. The fact that tectodendrimers behave differently suggests that their cluster-like architecture plays an important role in their interaction with the lipid bilayer.
... In small angle X-ray scattering spectra, clear widening distances between Bragg reflection peaks indicated the detachment of multilamellar from one another (possibly due to dendrimer insertion, [177]), while wide angle X-ray scattering spectra that correlate to the packing of aliphatic chains again showed minimal changes. The layer detachment is verified with freeze-fracture electron microscope, while small quantities of small vesicles/micelles are also observed (possibly the dendrimersomes hypothesized above [172]). This small population of spherical structures was assumed to be responsible for the appearance of a new peak in 31 P spectra. ...
... While imaging methods, for example AFM, are able to provide pictorial information of the interaction, the quantification of binding strength and physical changes of the lipid membrane during this binding event is beyond the capability of such approaches. [170][171][172] Our choice of technique to meet these requirements is therefore plasmon waveguide resonance (PWR) spectroscopy, a homemade modification of commonly known surface plasmon resonance (SPR) spectroscopy that specializes in lipid-molecule interaction analysis. onto the silica coating, meaning the analysis of material anisotropy can also be achieved by analyzing the PWR spectra. ...
... Parimi et al. studied the influence of PAMAM solution on substrate supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) lipid bilayer membrane with the combination of optical waveguide lightmode spectroscopy and AFM.[170] They found that regardless of dendrimer generation, at 1 nM and 10 nM concentrations, all the dendrimers were able to influence the amount of adsorbed lipid by aggravating the already existing defects (irregular in shape) on the supported lipid layer(Figure 18 A); while at100 nM all the dendrimers were able to remove materials from the adsorbed membrane by generating holes spontaneously(Figure 18 B).To study the mechanism of mass removal from the lipid bilayers, Mecke et al. also carried out similar experiments with AFM.[171,172] They proposed that PAMAM can interact with lipid membrane so strongly that it can rip part of the adsorbed lipid membrane from the substrate. ...
Microvesicles (MVs) have been a growing research interest at the front line of disease diagnosis. They are used by almost all types of cells in the human body as a tool of intercellular communication, thus can be sampled from most of the extracellular fluids without causing severe damage to the surrounding tissue. They are formed by budding of cell membrane, thus their membrane proteins are reminiscent to that of their parent cells, allowing them to be traced back to their parent cell types. They contain rich varieties of biomolecules including lipid, protein, and nucleic acids, thus the analysis of them will provide valuable physiological and pathological information on their cells of origin. They are released under cellular stress, thus they represent the early cellular response to corresponding stimuli. In other words, an analysis of them is able to provide an approach for the early detection of pathological conditions. A full characterization of the MVs includes their population per unit volume of the sample liquid, their size distribution, their morphology, their composition of lipid, protein, and nucleic acids. Unfortunately, there isn’t a platform developed allowing all the aforementioned tests at the same time, while existing test protocols are severely limited by the pre-analytical treatments, especially the purification processes. Herein, we present the design and construction of a device on which microvesicles can be captured, accommodating both physical and chemical analysis on the MVs within an extracellular fluid sample.For the device construction, we first synthesized a series of dendrons with increasing dendricity that are able to specifically bind to phosphatidylserine (PS), a lipid molecule exposing only on the outer leaflet of extracellular vesicles. The dendron peripherals are functionalized with dipicolylamine-Zn2+ (DPA-Zn) complexes to provide the dendron-phosphatidylserine interaction. The convergently synthesized dendrons are adopted as the molecular support for the complex units so that the binding ability of dendrons to PS can be controlled and improved through multivalency and synergy of neighboring DPA-Zn units. The core of each dendron is attached to an n-hexylamine spacer, allowing the attachment to the material surface.
... The structures around 1000 nm are probably composed of PAMAM G4 dendrimers with DDA:TDB lipids all around them. This is another possible mechanism for the formation of holes in the lipid bilayer [35]. from other groups and our previous studies [32][33][34]. ...
... This mechanism is the driving force for the formation of large aggregates with sizes around 1000 nm, according to cryo-TEM and DLS measurements. According to Mecke et al. 2004, the dendrimers sometimes remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules [35]. The structures around 1000 nm are probably composed of PAMAM G4 dendrimers with DDA:TDB lipids all around them. ...
... This mechanism is the driving force for the formation of large aggregates with sizes around 1000 nm, according to cryo-TEM and DLS measurements. According to Mecke et al. 2004, the dendrimers sometimes remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules [35]. The structures around 1000 nm are probably composed of PAMAM G4 dendrimers with DDA:TDB lipids all around them. ...
Liposomes with adjuvant properties are utilized to carry biomolecules, such as proteins, that are often sensitive to the stressful conditions of liposomal preparation processes. The aim of the present study is to use the aqueous heat method for the preparation of polymer-grafted hybrid liposomes without any additional technique for size reduction. Towards this scope, liposomes were prepared through the combination of two different lipids with adjuvant properties, namely dimethyldioctadecylammonium (DDA) and D-(+)-trehalose 6,6′-dibehenate (TDB) and the amphiphilic block copolymer poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) (PLMA-b-PDMAEMA). For comparison purposes, PAMAM dendrimer generation 4 (PAMAM G4) was also used. Preformulation studies were carried out by differential scanning calorimetry (DSC). The physicochemical characteristics of the prepared hybrid liposomes were evaluated by light scattering and their morphology was evaluated by cryo-TEM. Subsequently, in vitro nanotoxicity studies were performed. Protein-loading studies with bovine serum albumin were carried out to evaluate their encapsulation efficiency. According to the results, PDMAEMA-b-PLMA was successfully incorporated in the lipid bilayer, providing improved physicochemical and morphological characteristics and the ability to carry higher cargos of protein, compared to pure DDA:TDB liposomes, without affecting the biocompatibility profile. In conclusion, the aqueous heat method can be applied in polymer-grafted hybrid liposomes for protein delivery without further size-reduction processes.
... Установлено, что добавление нейтрального и анионного ПАМАМ дендримеров не влияло на вольт-амперные характеристики БЛМ. Добавление катионного дендримера приво- Ранее было показано, что взаимодействие катионного ПАМАМ дендримера 7-й генерации с липосомами может вызывать формирование пор [26][27][28], а введение катионного ПАМАМ дендримера 4-й генерации в смесь липидов при формировании модельных мембран может индуцировать нарушения в бислое, включая формирование гексагональной фазы [29]. В некоторых случаях катионные ПАМАМ дендримеры больших генераций (от 7-й и выше) были способны отрывать липиды от бислоя и формировать дендример-липидные везикулы [30], что могло привести к изменению проводимости липидного бислоя. ...
... Известно, что проводимость немодифицированной БЛМ определяется индуцируемыми метастабильными единичными порами с временем жизни ~3 мс [25] и размером пор ~0,6 нм [31]. Непосредственно перед срывом эти поры стабилизируются и увеличиваются в размерах, формируя ионные каналы [26][27][28]. В наших экспериментах в случае немодифицированных БЛМ появление метастабильных ионных пор наблюдалось в 32 % случаев. Добавление катионного дендримера приводило к выраженному снижению количества метастабильных одиночных пор, а при концентрации дендримера более 10 мкМ формирования ионных пор не наблюдалось. ...
... Таким образом, взаимодействие катионного дендримера с БЛМ приводит к резкому снижению сопротивления мембраны, исчезновению спонтанных одиночных пор и увеличению напряжения срыва мембраны вследствие встраивания дендримера в БЛМ. Обнаруженное в экспериментах снижение времени жизни БЛМ в присутствии дендримера также указывает на формирование агрегатов дендример-БЛМ, т. е. пор [26][27][28]. По-видимому, электростатические силы играют главную роль во взаимодействии дендример-БЛМ, поэтому нейтральный и анионный дендримеры не взаимодействуют с БЛМ (при рН выше 6,5 ФХ находится преимущественно в форме LOH -) [32,33]. ...
Polyamidoamine (PAMAM) dendrimers and amphiphilic dendrons are one of the types of nanomaterials characterized by a hyperbranched structure of polymer branches. In the case of dendrimers, the dendrons are covalently linked at the central focal point. In the case of amphiphilic dendrons, dendrons are non-covalently linked by hydrophobic interactions, forming micellar structures. These nanoparticles are widely used in biology and medicine as contrast agents, carriers of drugs and genetic material. Their use in scientific practice requires an understanding of the basic mechanisms of their interaction with membranes – the main obstacle to the entry of dendrimers into the cell. This review discusses the regularities of the interaction of dendrimers and amphiphilic dendrons with lipid membranes. Various models of dendrimer-membrane interactions are described as the basis for the penetration of dendrimers and amphiphilic nanoparticles into cells. Keywords: polyamidoamine dendrimers, amphiphilic dendrons, lipid membranes, cells, antitumor therapeutics, antibacterial agents, diagnostics, genetic therapy.
... [32][33][34] Notably, multiple studies have shown that commonly used cationic polymers (e.g., diethylaminoethyl (DEAE)-dextran, polyethyleneimine (PEI), polyamidoamine (PAMAM)-dendrimers) and cationic nanoparticles (e.g., surface-modified mesoporous silica nanoparticles and gold nanoparticles) can also induce membrane disruption events such as increased membrane fluidity, membrane thinning, and the formation of nanoscale holes, even at non-toxic concentrations. [34][35][36][37][38][39][40][41] Remarkably, studies using supported lipid bilayers as model membranes revealed that localized membrane thinning events typically precede the complete removal of lipids, [35] eventually resulting in membrane pores with an estimated average size of 15-40 nm in diameter. [37] In addition, theoretical studies have shown these pore formation events to be thermodynamically feasible. ...
... [34][35][36][37][38][39][40][41] Remarkably, studies using supported lipid bilayers as model membranes revealed that localized membrane thinning events typically precede the complete removal of lipids, [35] eventually resulting in membrane pores with an estimated average size of 15-40 nm in diameter. [37] In addition, theoretical studies have shown these pore formation events to be thermodynamically feasible. [35][36][37]42,43] Practical evidence for increased cell permeability was mainly provided by showing the influx of small membrane-impermeable molecules (e.g., fluorescent dyes and ions) in viable, polycation-treated cells. ...
... [37] In addition, theoretical studies have shown these pore formation events to be thermodynamically feasible. [35][36][37]42,43] Practical evidence for increased cell permeability was mainly provided by showing the influx of small membrane-impermeable molecules (e.g., fluorescent dyes and ions) in viable, polycation-treated cells. [34,36,43,44] Given the aforementioned studies, we hypothesized that cationic polymers and nanoparticles could not only function as carrier-mediated delivery devices, but could also be positioned as membrane-disruptive agents to enable direct cytosolic delivery of membrane-impermeable compounds. ...
Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)–dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1-weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.
... Supported lipid bilayers (SLBs) are well suited for studying the dendrimer-membrane interactions, as they lend themselves readily to quantitative analysis using a range of surface sensitive techniques such as X-ray and neutron reflectivity (XRR and NR) [14,80,81], atomic force microscopy (AFM) [82][83][84], ellipsometry [14,85], quartz crystal microbalance with dissipation (QCM-D) [81,85,86], NMR [87], differential scanning calorimetry (DSC) and Raman spectroscopy [88]. ...
... It was found that an increase in the dendrimer concentration caused the existing defects to expand and encouraged formation of new holes in the bilayers. Lipids removed from the bilayer in the hole formation wrapped around the G6 dendrimers, forming aggregates (dendrisomes) in solution, some of which adsorbed to the exposed substrate, also observed by Mecke et al. [84,96]. As described by several other AFM studies [65,84,96,99], higher generation (G4 and G6) dendrimers caused lipid desorption to a larger extent, compared to lower generation (G2) dendrimers, due to their increased size and charge density. ...
... Lipids removed from the bilayer in the hole formation wrapped around the G6 dendrimers, forming aggregates (dendrisomes) in solution, some of which adsorbed to the exposed substrate, also observed by Mecke et al. [84,96]. As described by several other AFM studies [65,84,96,99], higher generation (G4 and G6) dendrimers caused lipid desorption to a larger extent, compared to lower generation (G2) dendrimers, due to their increased size and charge density. Hong et al. [65] and Mecke et al. [84,96] used similar systems to study the effect of dendrimer surface functional groups on their interactions with DMPC SLBs. ...
PAMAM dendrimers have been conjectured for a wide range of biomedical applications due to their tuneable physicochemical properties. However, their application has been hindered by uncertainties in their cytotoxicity, which is influenced by dendrimer generation (i.e. size and surface group density), surface chemistry, and dosage, as well as cell specificity. In this review, biomedical applications of polyamidoamine (PAMAM) dendrimers and some related cytotoxicity studies are first outlined. Alongside these in vitro experiments, lipid membranes such as supported lipid bilayers (SLBs), liposomes, and Langmuir monolayers have been used as cell membrane models to study PAMAM dendrimer-membrane interactions. Related experimental and theoretical studies are summarized, and the physical insights from these studies are discussed to shed light on the fundamental understanding of PAMAM dendrimer-cell membrane interactions. We conclude with a summary of some questions that call for further investigations.
... Moreover, many publications show correlations between results obtained with model membranes and in vitro cell studies. [3][4][5][6][7] Dendrimer-lipid model membrane interactions of many kinds of dendrimers with different cores, surface modifications and generations have been investigated, and different effects have been observed. Dendrimers are known to be able to pass through the membrane, incorporate into the lipid bilayer or remove some lipids and create dendrimer-lipid micelles. ...
... Interestingly, investigations have evidenced that although low-generation anionic dendrimers induce liposome disruption, higher-generation (G5) anionic dendrimers are able to induce lipid packing in the bilayer structure. 3,4 Some investigations also suggest that it is possible to observe different types of interaction according to the dendrimer concentration used. It has been shown that PAMAM dendrimers at a low concentration can bind to the membrane surface and then, due to their flexible structure, traverse the bilayer and dissociate from the membrane on the other side. ...
... 17,18 Here, we report the study of dendrimer-lipid membrane interactions with recently introduced novel types of 1 st -3 rd -generation carbosilane dendrimers. 19 Dendrimers with the five different types of terminal phosphonium groups (PMe 3 , PBu 3 , P(Et 2 ) 2 (CH 2 ) 3 OH, PPh 3 and P(MeOPh) 3 ) and one type of ammonium terminated carbosilane dendrimer (NMe 3 ) have been studied. Ammoniumterminated dendrimers were used for a comparison to the new types of phosphonium-terminated ones. ...
The influence of three generations of five different phosphonium carbosilane dendrimers and one ammonium carbosilane dendrimer as a reference (PMe3, PBu3, P(Et2)2(CH2)3OH, PPh3, P(MeOPh)3 and NMe3, peripheral functional groups) on dimyristoylphosphatidylcholine (DMPC) or a lipid mixture dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) of liposomes was studied by fluorescence polarization measurements and differential scanning calorimetry. All types of dendrimers interacted with neutral as well as negatively charged liposomes, but the strength and observed influence were different. Concentration, type of peripheral functional group modification and dendrimer generation were the main factors influencing the interaction. Generally, weak interactions as well as destabilization of the lipid membranes at low concentrations, regardless of liposome type, were observed in the case of DmPMe3, DmNMe3, DmPBu3 and DmP(Et2)2(CH2)3OH. Dendrimers with PPh3 and P(MeOPh)3 peripheral functional groups interacted much more strongly and increased the rigidity of liposomes. Electrostatic interactions, the hydrophobicity of substituents as well as charge shielding on the peripheral phosphonium group are important factors in the interaction. We suggest that, among the other types of dendrimers, the dendrimer with the P(MeOPh)3 peripheral functional group is a highly promising candidate for the design of a drug delivery system due to its positive charge, efficient interaction with lipidic membranes and low cytotoxicity.
... The toxicity of dendrimers is an important consideration in their synthesis and testing, and higher-generation dendrimers often confer greater toxicity compared to lower-generation dendrimers [83]. Dendrimers with cationic charges, such as PAMAM and PPI dendrimers, can interact with and destabilize cellular membranes, producing nanoscale holes in the lipid membranes that allow leakage of material and increase membrane permeability [92][93][94]. Pryor et al. illustrated that G6-amine PAMAM dendrimers were significantly more toxic than neutral and anionic PAMAM dendrimers at the same concentration in embryonic zebrafish, illustrating the important influence of surface charges. Toxicity was measured by assessing mortality, developmental progression, and physical malformations [95]. ...
... The toxicity of dendrimers is an important consideration in their synthesis and testing, and higher-generation dendrimers often confer greater toxicity compared to lowergeneration dendrimers [83]. Dendrimers with cationic charges, such as PAMAM and PPI dendrimers, can interact with and destabilize cellular membranes, producing nanoscale holes in the lipid membranes that allow leakage of material and increase membrane permeability [92][93][94]. Pryor et al. illustrated that G6-amine PAMAM dendrimers were signif- Additionally, cationic dendrimers have been associated with a pro-coagulant role and can induce platelet aggregation in vivo, resulting in a coagulation cascade that can produce a syndrome similar to disseminated intravascular coagulation. Jones et al., in a study also using embryonic zebrafish, illustrated that G7 PAMAM dendrimers can interact with a variety of blood components, including platelets, fibrinogen, and anionic blood proteins, producing a unique, multi-component, rapid coagulation cascade. ...
Simple Summary
Gliomas are common primary brain tumors that account for a high number of cancer-related deaths worldwide. Patients with aggressive gliomas have poor prognoses due to resistance to current treatment regimens. Anatomical barriers between the brain and the peripheral circulation make glioma drug delivery particularly challenging. Recently, the potential for dendrimers—nanomaterials characterized by a unique branching structure with many binding sites—to deliver therapeutic and diagnostic cargo directly to the site of gliomas has been explored. An increasing number of studies in glioma cell lines and animal models suggest that this dendrimer-based delivery approach may serve to decrease drug toxicity and allow for the use of nontraditional therapeutic and imaging agents in glioma. Here, we review the physical characteristics of dendrimers, strategies for modifying dendrimers to target glioma cells, and novel applications of this nanotechnology in glioma models.
Abstract
Novel therapeutic and diagnostic methods are sorely needed for gliomas, which contribute yearly to hundreds of thousands of cancer deaths worldwide. Despite the outpouring of research efforts and funding aimed at improving clinical outcomes for patients with glioma, the prognosis for high-grade glioma, and especially glioblastoma, remains dire. One of the greatest obstacles to improving treatment efficacy and destroying cancer cells is the safe delivery of chemotherapeutic drugs and biologics to the tumor site at a high enough dose to be effective. Over the past few decades, a burst of research has leveraged nanotechnology to overcome this obstacle. There has been a renewed interest in adapting previously understudied dendrimer nanocarriers for this task. Dendrimers are small, highly modifiable, branched structures featuring binding sites for a variety of drugs and ligands. Recent studies have demonstrated the potential for dendrimers and dendrimer conjugates to effectively shuttle therapeutic cargo to the correct tumor location, permeate the tumor, and promote apoptosis of tumor cells while minimizing systemic toxicity and damage to surrounding healthy brain tissue. This review provides a primer on the properties of dendrimers; outlines the mechanisms by which they can target delivery of substances to the site of brain pathology; and delves into current trends in the application of dendrimers to drug and gene delivery, and diagnostic imaging, in glioma. Finally, future directions for translating these in vitro and in vivo findings to the clinic are discussed.
... The issue of the mass transfer limitations could be further understood by the interaction of particles with phospholipid membranes [27][28][29]. The artificial membrane was disturbed or destroyed to varying degrees by different physicochemical properties of microparticles [30][31][32], especially the size of the particles [33][34][35]. Meanwhile, the ability of the particles to pass through the membrane was also affected by the surface chemical modification of nanoparticles [36]. ...
... As shown in Figure 3, the mass transfer coefficient of that was plotted versus transfer times to investigate the transmembrane transmission of different substances. to varying degrees by different physicochemical properties of microparticles [30][31][32], especially the size of the particles [33][34][35]. Meanwhile, the ability of the particles to pass through the membrane was also affected by the surface chemical modification of nanoparticles [36]. ...
Microbial pigments, regarded as the most potential biomass pigments, have lately attracted increasing attention in textile dyeing due to their sustainability and cleaner production. The pyrrole structure microbial pigment, called prodigiosin, recently have become a research hotspot for its bright colors and antibacterial function. However, in most case the extraction and preparation are time-consuming and expensive processes since these kinds of microbial pigments are intracellular metabolites. In order to promote the application of microbial pigments in textile dyeing, a novel idea of preparing dye liquid of pyrrole structure pigments based on fermentation broth was put forward via increasing the proportion of extracellular pigments. A model membrane platform was established with a planar lipid bilayer to investigate transmembrane transport of microbial pigments and permeability barrier of cell membrane. The nano-dispersion of pigments was produced as the dye liquor owing to high-throughput transmembrane transfer of intracellular pigments and the increase of extracellular pigments proportion. The results indicated that the size and surface electrical properties of the pigments had contributed much to the mass transfer. It is also showed that transmembrane transmission of the intracellular pigments could be regulated by physical and chemical methods. With the improvement of transmembrane transfer efficiency of microbial pigments and the proportion of extracellular pigments, the complicated biological separation process could be avoided and the application of microbial pigments in textile dyeing can be promoted.
... [177][178][179] Different degrees of membrane damages like membrane thinning due to lipid reorientation or lipid removal, and transient membrane hole formation were identified, which was influenced by the polymer molar mass, charge density and concentration. 176,[179][180][181] Interestingly, nanoscale membrane holes induced the leakage of intracellular molecules or dyes, 178,179 but did neither facilitate the cellular entry of polyplexes nor gene expression. 182 Since autophagy can be triggered by endosomal membrane damage, 183,184 a direct evidence for polyplex-mediated membrane disruption or nanoscale hole formation could be given by the detection of autophagy markers. ...
... Polymer-membrane interaction of lipid bilayers by AFM. [178][179][180] (II) Charge-driven interaction of polyplex with endolysosomal membrane. ...
The targeted and efficiency-oriented delivery of (therapeutic) nucleic acids raises hopes for successful gene therapy for the local and individual treatment of acquired and inherited genetic disorders. Despite promising achievements in the field of polymer-mediated gene delivery, the efficiency of the non-viral vectors remains orders of magnitude lower than viral-mediated ones. Several obstacles on the molecular and cellular level along the gene delivery process were identified, starting from the design and formulation of the nano-sized carriers up to the targeted release to their site of action. In particular, the efficient escape from endo-lysosomal compartments was demonstrated to be a major barrier and its exact mechanism still remains unclear. Different theories of the endosomal escape were postulated. The most popular one is the so-called “proton sponge” hypothesis, claiming an escape by rupture of the endosome through osmotic swelling. It was the first effort to explain the excellent transfection efficiency of poly(ethylene imine). Moreover, it was thought that a unique mechanism based on the ability to capture protons and therefore to buffer the endosomal pH is the basis of escape. Recent hypotheses deal with the direct interaction of the cationic polyplex or free polymer with the exoplasmic lipid leaflet causing membrane destabilization, permeability or polymer-supported pore formation. Both escape strategies are more related to viral-mediated escape compared to the “proton sponge” effect, hence, it seems that polycations are able to use a mechanism that viruses exploit since centuries. This review addresses the different endosomal release theories that have been hypothesized and highlights their key mechanism. Additionally, the validity of each theory is discussed in detail.
... Using coarse-grained (CG) MARTINI models, Lee and Larson 37,40−42 showed that higher generation (G7, G5) PAMAM dendrimers can form pores in lipid membranes, and the pore formation efficacy is reduced for acetylated and PEGylated dendrimers in agreement with experimental observations. 27,43 The nature of dendrimer interactions with lipid membranes strongly depends on the pH of the solvent. Using a MARTINI model, dendrimers were found to have an attractive interaction with negatively charged mixed (DPPC/ DOPG) lipid membranes and the free energy of binding is negative at low pH whereas the interaction becomes repulsive at high pH. ...
... Additionally, these simulations were carried out for a circular patch of lipids with a cylindrical hard wall imposed to constrain the area. Despite these simplifications, the results qualitatively agree with experimental data of Mecke et al., 26,43,45,46 which reveal binding of G3 PAMAM dendrimers on DMPC membranes. Area fluctuations and lipid mobility play an important role during binding of proteins and polymers to membranes 47−49 and are expected to influence the final state of the dendrimer-membrane complex. ...
... [49,50] The molecular weight, charge density, and concentration of the polymer remarkably affected the degree of membrane damage, including membrane thinning due to lipid removal or reorientation and transient membrane hole formation. [50,51] Interestingly, nanoscale membrane pores caused intracellular molecules or dyes to leak, but they did not affect how polyplexes entered the cells or how genes were expressed. [49,50] Several studies used fluorophore-tagged microtubule-associated protein and Galectin-8/Galectin-9 markers to investigate endosomal escape and the membrane damage caused by nonviral gene delivery vectors in real-time by live cell imaging. ...
Nucleic acid‐based therapies are seeing a spiralling surge. Stimuli‐responsive polymers, especially pH‐responsive ones, are gaining widespread attention because of their ability to efficiently deliver nucleic acids. These polymers can be synthesized and modified according to target requirements, such as delivery sites and the nature of nucleic acids. In this regard, the endosomal escape mechanism of polymer–nucleic acid complexes (polyplexes) remains a topic of considerable interest owing to various plausible escape mechanisms. This review describes current progress in the endosomal escape mechanism of polyplexes and state‐of‐the‐art chemical designs for pH‐responsive polymers. We also discuss the importance of the acid dissociation constant (i.e., p K a ) in designing the new generation of pH‐responsive polymers, along with assays to monitor and quantify the endosomal escape behavior. Further, we address the use of machine learning in p K a prediction and polymer design to find novel chemical structures for pH responsiveness. This review will facilitate the design of new pH‐responsive polymers for advanced and efficient nucleic acid delivery.
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... One of the possible mechanisms of the damaging effect of nanoparticles at the molecular level can be their effect on the tertiary structure of proteins. It may be accompanied by immune reactions towards denatured proteins that acquire the properties of antigens alien to the body, by increased proteolysis, apoptosis, necrosis, inflammation, and membrane permeability [144,145]. Fractions of histone proteins and heat-shock proteins are informative indicators of changes in cells at an early stage of nanoparticles influence before the appearance of the "macrotoxicity" signs. The synthesis of inflammatory factors by lymphocytes depends on the modifications and the amount of histone proteins, the release of which from DNA is essential for the expression of proinflammatory factor genes [146][147][148]. ...
The paper discusses the results of a research on physicochemical and biochemical properties of the Keplerate-type molybdenum-based nanocluster polyoxometalates (POMs), which show promise in the field of biomedicine as a means of targeted drug delivery, including the transport to immune privileged organs. POMs can be considered as components of releasing systems, including the long-acting ones with feedback (for controlling the drug active component release rate). POMs are promising drugs for the treatment of anemia. Also, the paper deals with the results of studies of POM effect on living systems at the molecular and cellular levels, at that of individual organs, and on the organism as a whole. The mechanism and kinetics of POM destruction and possibilities of stabilization, the oscillatory phenomena manifestation, the formation of POM conjugates with bioactive substances which can be released during the destruction of POM, with polymer components, and with indicator fluorescent dyes, as well as forecasts for further research, are considered.
... In many studies, cationic polymers have been shown to exhibit high transfection efficiencies at relatively high weight ratios. This phenomenon occurs because free polymers combined with plasmids promote cellular uptake and endosomal escape by forming nanosized holes in the cellular membrane and inducing osmotic swelling and bursting [24,25]. Based on our physical property analysis, we concluded that the PAMAM derivative may exhibit optimal transfection efficiency at ratios ranging from 4:1 to 16:1 (w/w). ...
Polyamidoamine (PAMAM) dendrimers are biocompatible polymers utilized in multiple biomedical applications including tissue engineering, medical diagnosis, drug and gene delivery systems, and biosensors. Normally, high-generation PAMAM dendrimers are advantageous for use in gene therapy research because they have a relatively high transfection efficiency. A high-generation PAMAM dendrimer has a high charge density, which induces greater damage to the membranous organelles than that induced by a low-generation PAMAM dendrimer. In this study, we added NLS sequences derived from the human papillomavirus (HPV) type 11 E2 protein to the low-generation PAMAM generation 2 (PAMAM G2) dendrimer and simultaneously introduced histidine residues to reduce cytotoxicity. RKRARH-PAMAM G2 showed similar and high transfection efficiencies in Neuro-2A and NIH3T3 cell lines and relatively low cytotoxicities relative to that of polyethylenimine 25 kDa (PEI 25kDa).
... Various forms of designer and functional nanoparticles (NPs) have been developed as nanoprobes for various biomedical applications including bioimaging probes, drug delivery carriers and theranostics among others. [1][2][3][4][5][6][7][8][9][10] In particular, fluorescent NPs like quantum dots (QDs) having high quantum efficiency are very suitable for in vitro and small animal imaging in general and single molecule imaging applications in complex bioenvironments in particular. It is also well known that the cell-nanoparticle interaction is highly sensitive to the NP surface chemistry and surface charge [11][12][13][14][15][16][17] and hence the nanoprobe surface must be appropriately designed for the purpose for which it is meant to be used. ...
Designing of nanoparticles (NPs) for biomedical applications or mitigating their cytotoxic effects require microscopic understanding of their interactions with cell membranes. Such insight is best obtained by studying model biomembranes which, however, need to replicate actual cell membranes, especially its compositional heterogeneity and charge. In this work we have investigated the role of lipid charge density and packing of phase separated Langmuir monolayers on penetration and phase specificity of charged quantum dot (QD) binding. Using an ordered and anionic charged lipid in combination with uncharged but variable stiffness lipids we demonstrate how subtle interplay of zwitterionic lipid packing and anionic lipid charge density can affect cationic nanoparticle penetration and phase specific binding. Under identical subphase pH, the membrane with higher anionic charge density displays higher NP penetration. We also observe coalescence of charged lipid rafts floating amidst a more fluidic zwitterionic lipid matrix due to phase specificity of QD binding. Our results suggest effective strategies which can be used to design NPs for diverse biomedical applications as well as in devising remedial actions against their harmful cytotoxic effects especially against respiratory diseases.
... By contrast to charged PECs, a much larger concentration of neutral PECs was required to induce such defects, and the polymer could not only intercalate in the hydrophobic tail region but also spread on top of the bilayer. 11 Besides these interactions involving the cationic vectors, the presence of the genome in the membrane-bound PEC induced alteration in the molecular order of the bilayer since systematic studies of the complexation of DNA with liposomes provided evidence for intricate interactions. 12,13 On one hand, the surface-bound genome stabilized the membrane through electrostatic interactions with the polar headgroup, and on the other hand, when DNA penetrated the lipid hydrophobic part of mixed membrane, the results led to microdomain lipid segregation and lipid long-range molecular order. ...
Neutral polyelectrolyte complexes (PECs) made from an amphiphilic multiblock copolymer of type (AnBn)m and an oppositely charged polyion and interacting with a dipalmitoylphosphatidylcholine (DPPC) lipid membrane have been examined employing a coarse-grained model with implicit solvent and molecular dynamics simulations. One systematically explored the influence of the size of the hydrophobic block B and of the number of these blocks per chain on the PEC tendency to adhere to the membrane surface and to intercalate into the membrane core. Simulation results showed that PECs bound irreversibly to the lipid bilayer without polyion unwinding from the complex and the adsorbed conformation was strongly affected by the size of the hydrophobic block B. The adsorption kinetics at low B size were characterized by a relaxation phase dominated by the spreading of PEC constituents along the outer leaflet of the membrane. Upon increasing the size of the hydrophobic block B to reach core–shell organization of the free PEC, the relaxation pathway of the complex corona in close contact with the headgroup lipids facilitated the transient exposure of the PEC hydrophobic core to the lipids and its subsequent cooperative internalization and solubilization in the membrane inner part associated with an internal reorganization of the lipid bilayer. In the generated snorkeling-type conformation, the charged blocks A and the oppositely charged polyion were confined to the headgroup region of the top leaflet, without spontaneous flipping to the headgroup region of the distal leaflet.
... Figure 14 exhibits the morphologies of cell exposed to a series of concentration from 0.1 to 100 μg/mL by fluorescence image. We clearly observe the aggregation of dead cell at P4′ concentration of 100 μg/mL as compared to P4. e higher cytotoxicity results from the fact that positive charge of P4′ side chains can disrupt lipid bilayers and finally kill cell [2,30]. To ensure materials uptake by cells, low cytotoxicity is substantially valuable [31]. ...
Cationic and zwitterionic polyelectrolytes are synthesized through atom transfer radical polymerization (ATRP), comprising a polyfluorene backbone with a small fraction of 2,1,3-benzothiadiazole and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) side chains. Due to higher charge density generated from grafted side chains, two polymers show higher water solubility and higher quantum yield. In comparison with cationic polyelectrolytes, zwitterionic polyelectrolytes are stable over a broad pH range from 1 to 13, even in 1 M NaCl solution. The absence of FRET between zwitterionic polymers and dye-labeled ssDNA indicates their ultralow nonspecific adsorption, while cationic polymer shows much stronger nonspecific interactions. The MTT assay of zwitterionic polymers exhibits their minimal cytotoxicity and potential in long-term clinical application. Most importantly, zwitterionic polymer could be efficiently taken up by cells, whereas cationic polymer stains the surface of cell due to membrane disruption generated from positive charges. The results illustrate that conjugated zwitterionic polymer could serve as a novel type of highly efficient ultralow fouling material with low cytotoxicity for labelling cell or potential biomedical applications.
... 7,22 They interact with negatively charged lipids and disrupt the lipid bilayer and can induce hole formation in model lipid bilayers and cell membranes. [22][23][24][25][26] The resulting holes can be related to intracellular transport and the passive diffusion of large particles that usually must be actively transported into and out of the cell. PAMAM dendrimers' ability to disrupt cellular membranes is dependent on their size and the composition of end groups. ...
... A number of classes of compounds are known to disrupt cell membranes by interfering with bilayer structure, including dendrimers [1], chitosan [2], and membrane-active lytic peptides [3]. In this review, the focus is on the disruption of membrane structure by the class of compounds known as glycoalkaloids [4,5] which exert such effects by binding to cholesterol that is ubiquitously present in cell membranes [6], forming complexes that can aggregate and compromise the membrane structure. ...
The glycoalkaloids which are secondary metabolites from plants have proven to be of significant interest for their biological properties both in terms of their roles in plant biology and the effects they exhibit when ingested by humans. The main feature of the action of glycoalkaloids is their strong binding to 3β-hydroxysterols, such as cholesterol, to form complexes with the consequence that membrane structure is significantly perturbed, and leakage or release of contents inside cells or liposomes becomes possible. The glycoalkaloids have been studied for their ability to inhibit the growth of cancer cells and in other roles such as vaccine adjuvants and as synergistic agents when combined with other therapeutics. The glycoalkaloids have rich and complex physical behavior when interacting with model membranes for which many aspects are yet to be understood. This review introduces the general properties of glycoalkaloids and aspects of their behavior, and then summarizes their effects against model membrane systems. While there are many glycoalkaloids that have been identified, most physical or biological studies have focused on the readily available ones from tomatoes (α-tomatine), potatoes (α-chaconine and α-solanine), and eggplant (α-solamargine and α-solasonine).
... Cytotoxicity of PAMAM dendrimers in Caco-2 cell lines [106] and cytotoxicity of PPI and PAMAM dendrimers in B16F10, CCRF, and HepG2 cancer cell lines [107] have been reported. Numerous studies have examined that cationic dendrimers interact with negatively charged biological membrane to produce nanoscale holes and cell lysis [108][109][110][111][112]. The mechanism of cationic PAMAM dendrimer induced membrane damage using 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers, and KB and Rat2 cells in culture have been reported by Hong et al. [113]. ...
Since the development of first lipid-based nanocarrier system, about 15% of the present pharmaceutical market uses nanomedicines to achieve medical benefits. Nanotechnology is an advanced area to meliorate the delivery of compounds for improved medical diagnosis and curing disease. Nanomedicines are gaining significant interest due to the ultra small size and large surface area to mass ratio. In this review, we discuss the potential of nanotechnology in delivering of active moieties for the disease therapy including their toxicity evidences. This communication will help the formulation scientists in understanding and exploring the new aspects of nanotechnology in the field of nanomedicine.
... Drugs can be complexed with dendrimers through encapsulation into void spaces (nanoscale container), association with the surface groups (nano-scaffolding), or both [6,50]. The high density of surface groups (one amino group/nm 2 for DG4) combined with the small size (4.5 nm diameter for the DG4 ellipsoids) result in a high area/ volume ratio [51,52], which can be modified controlling the environment ionic strength, pH, temperature, etc. ...
... However, dendrimers with high generations results in higher toxicity due to a large number of terminal cationic groups which can interact with negatively charged cell components, e.g. cell membranes causing their disruption [101,102]. This disadvantage can be diminishing by surface modification with different targeting or shielding moieties providing with not only low toxicity but also enhance the cell uptake and specific accumulation of nucleic acid molecules inside cells [103][104][105][106]. ...
Synthetic polymers, such as dendrimers, play a critical role in pharmaceutical discovery
and development. Advances in the application of nanotechnology in medicine have given
rise to multifunctional “smart” nanocarriers that can deliver one or more therapeutic
agents safely and selectively to cancer cells, including intracellular gene-speciic targeting.
Dendrimers with their 3D nanopolymeric architectures are highly atractive class of
drug and gene delivery vector. Advances in understanding and manipulating genes gave
scientists a tool to make changes in people DNA to prevent or treat diseases. Over the past
decade, gene therapy has been in use in clinical trials. The inactivation of the tumor suppressor
genes is the main idea of the development of gene therapy in the cancer treatment.
Broad spectrum of delivery concepts, including viral vectors, liposomes, cationic polymers
and dendrimers, cell-penetrating peptides and gold and magnetic nanoparticles have been
investigated. A well-designed vector is the most desirable approach to increase the safety
of gene therapy, which is still in its infancy stages in cancer research. More experimental
and clinical trials are focused on well-designed and efective doses of vectors that are
essential for therapeutic eicacy of gene therapy for its potential in clinical use against a
wide variety of cancers.
... One of the most important limits is that most of the commonly used dendrimers are nondegradable in physiological environment, which will result in serious side effects induced by the accumulation of nondegradable artificial macromolecules inside cells or in tissues [21][22][23]. For instance, dendrimers like PAMAM and PPI not only exert remarkable in vitro cytotoxicity due to their surface cationic groups, but also associate with cell membrane disruption and succedent necrosis/non-apoptotic cell death [24][25][26][27][28][29][30][31]. In recent years, researchers have tried some strategies to keep away from the toxicity of dendrimers, such as crosslinking low generation dendrimers with biodegradable linkers [32,33]. ...
Nanoscience has become a worldwide multidisciplinary area on the basis of its unique physical and chemical traits, which have improved over the last few years. The atypical traits have given rise to diverse application in various domains of life. Solid particles are nanoparticles that have a dimension limit of 1–100 nm. Different sources are used to obtain nanoparticles via artificial synthesis using top-down and bottom-up processes and the environment via dust storms, anthropogenic, volcanic ash, and other natural processes. Lately, natural biogenic nanoparticles have become a popular subject matter due to their environmental and health gain. Nanomaterials can be created in natural world by employing a range of microorganisms, aquatic sources, and plants and can be produced in the laboratory. Biosynthesis of nanoparticles via plant- and microbe-mediated processes is an ecofriendly substitute to the high-cost, laborious, and probably toxic chemical and physical production method. This section discusses the different types of nanoparticles and their artificial and natural origins as well as categorization and biological synthesis using microbes, plants, and artificial procedures.
Nanotechnology and nanoengineering tend to generate significant scientific and technological push on in diverse domain including medicine and physiology. Nanodevices are acceptable to serve as customized, targeted drug delivery vehicles to convey large doses of chemotherapeutic agents or therapeutic genes into poisonous cells while cautious healthy cells. The considerable immediate influence of nanotechnologies in cancer remedy is in drug delivery. The therapeutic index of about all drugs presently being used can be upgraded if they are more ably delivered to their biological targets across appropriate application of nanotechnologies. Some drugs that have earlier failed clinical trials might also be reinvestigated using nanotechnological techniques. A number of hurdles may be overcome with numerous novel approaches of nano drug delivery. Drug-loaded nanoparticles are able to perforate this barrier, and have been appear to greatly increase therapeutic concentrations of anticancer drugs in brain tumors. Various applications of nanotechnology are biomedical nanodevice, disinfection, diagnosis, bionanosensor, drug screening, controlled drug delivery, drug targeting, tissue engineering, advanced medical imaging, etc. Nanoparticles are classified as one-dimensional nanoparticle, two-dimensional nanoparticles, and three-dimensional nanoparticle. Nanoparticles are prepared by emulsion-solvent evaporation method, salting-out method, emulsions diffusion method, and solvent- displacement/precipitation method. Nanoparticles are characterized by particle size (DLS, SEM & TEM used for determination of particle size), surface charge, and surface hydrophobicity. Advantages of nanostructure-moderated drug delivery comprise the capacity to deliver drug molecules directly into cells and the capacity to target tumors within healthy tissue. Nanoscale drug delivery architectures are able to puncture tumors due to the discontinuous, or “porous,” nature of the tumor microvasculature, which usually contains pores ranging from 100 to 1000 nm in diameter. The best means to expand the potency and reduce the toxicity of a cancer drug is to straight the drug to its target and continue its concentration at the site for an adequate time for therapeutic activity to take outcome. As the therapeutic segment of the nanocomplex, a mutant RAF gene was integrated to the particle for transfection and apprehension in the tumor cells. A novel approach for this technology is to use oligonucleotides for sensitizing tumor cells to chemotherapy. The oligonucleotides are being combined with nanoliposomes to target and deliver the nucleic acids to the cancer cells and block production of the alpha folate receptor. Various plants are used for the treatment of cancer, some of them are Aglaia roxburghiana Miq. Hiern, Bauhinia racemosa Lamk, Camellia sinensis (Linn.) O. Kuntze., Corchorus aestuans Linn, etc.KeywordsNanoparticlesTargetingTumor cellsNanotechnologyLiposomes
Amphiphilic dendrons represent a relatively novel class of molecules which may show many unique properties suitable for applications in a field of molecular biology and nanomedicine. They were frequently studied as platforms suitable for drug delivery systems as were, e.g. polymersomes or hybrid lipid-polymer nanoparticles. Recently, natural extracellular lipid vesicles (EVs), called exosomes (EXs), were shown to be a promising candidate in drug delivery applications. Formation of hybrid exosome-dendron nanovesicles could bring benefits in their simple conjugation with selective targeting moieties. Unfortunately, the complex architecture of biological membranes, EXs included, makes obstacles in elucidating the important parameters and mechanisms of interaction with the artificial amphiphilic structures. The aim of the presented work was to study the interaction of two types of amphiphilic carbosilane dendritic structures (denoted as DDN-1 and DDN-2) suitable for further modification with streptavidin (DDN-1) or using click-chemistry approach (DDN-2), with selected neutral and negatively charged lipid model membranes, partially mimicking the basic properties of natural EXs biomembranes. To meet the goal, a number of biophysical methods were used for determination of the degree and mechanisms of the interaction. The results showed that the strength of interactions of amphiphilic dendrons with liposomes was related with surface charge of liposomes. Several steps of interactions were disclosed. The initialization step was mainly coupled with amphiphilic dendrons - liposomes surface interaction resulting in destabilization of large self-assembled amphiphilic dendrons structures. Such destabilization was more significant with liposomes of higher negative charge. With increasing concentration of amphiphilic dendrons in a solution the interactions were taking place also in the hydrophobic part of bilayer. Further increase of nanoparticle concentration resulted in a gradual dendritic cluster formation in a lipid bilayer structure. Due to high affinity of amphiphilic dendrons to model lipid bilayers the conclusion can be drawn that they represent promising platforms also for decoration of exosomes or other kinds of natural lipid vehicles. Such organized hybrid dendron-lipid biomembranes may be advantageous for their subsequent post-functionalization with small molecules, large biomacromolecules or polymers suitable for targeted drug-delivery or theranostic applications.
Polyamidoamine (PAMAM) dendrimer has received much attention as an alternative to polyethylenimine (PEI) for gene delivery due to the relatively low cytotoxicity. In general, low generational PAMAM dendrimers have better biocompatibility than high generational dendrimers but suffer reduced transfection efficiency. Transfection efficiency can be improved by the modification of the polymer with nuclear localization signal (NLS) peptides. In this study, we modified low generational cystamine core PAMAM dendrimers (cPAMAM, generation 0, 1 and 2) with a lactoferrin-derived nuclear localization signal (NLS) peptide and evaluated transfection efficiency and cytotoxicity as a function of the number of conjugated NLS peptides using NIH 3T3, MCF-7 and human dermal fibroblasts (HDFs). The transfection efficiency of NLS-modified cPAMAM G2 was the highest among the cPAMAM derivatives and similar or higher than PEI 25 kDa. The cytotoxicity of cPAMAM derivatives was generation-dependent and significantly lower than PEI 25 kDa. Our study indicates that cPAMAM G2 conjugated with NLS is a promising candidate for gene delivery applications.
Nanotechnology is the science that deals with study of creation and utilization of nanoparticle. The size of nanoparticles are ranges from 1nm to 100 nm with one or more dimensions. There are various current trends regarding the application of nanoparticles like Herbal nanoparticles, Periodontics. Dental implants, Bionanoparticles, Gene delivery etc. some other trends of nanoparticles i.e. cancer treatment, drug delivery, cosmetics, agriculture application, which are widely applicable. The nanoparticles shows various aspects depending upon their size, composition, and structure along with their self-organised film structure. Nanotechnology plays a important role in our life. Almost every industry in recent scenario, nanoparticles or nanotechnology has been widely used. Nanotechnology have various field of application such as nanoclays, nanocomposites, nanocoating, nanoelectronics, nanobiotechnology, DNA replication, food, cosmetics as well as healthscience and medical sciences. In this review, the recent and current status of nanotechnology has been focused on the basis of various applications and result based on natural aspects.
The electrostatic interactions between cationic poly(amidoamine) (PAMAM) dendrimers of different generations, G3, G4, and G6, with net anionic model biomembranes have been predicted by adopting an analytical model based on two dissimilar soft spheres. The influence of bilayer surface charge density, ionic strength, pH, temperature, membrane softness (modeled as changes in bilayer thickness), and dendrimer generation on the attractive interaction was investigated. The attraction was found to decrease with increasing salt concentration, dendrimer charge, and thickness (or softness) of the membrane. On the other hand, the attraction increased with the surface charge density of the membrane, and the size of dendrimer generation. In fact, the attraction was found to be much larger for large generations, like G6 dendrimer that have a higher charge, than it is with small ones like G3 and G4 dendrimers. These results have implications for the use of PAMAM dendrimers as potential gene transfection vectors.
Nanoparticles can offer important advantages over the administration of conventional drugs in terms of high stability, high specificity, high drug transport capacity, controlled release capacity, possibility of use in different routes of administration and the ability to administer drugs both hydrophilic and hydrophobic molecules. Nanoparticles are being used for various purposes, from medical treatments, use in various branches of the production industry such as solar and for energy storage, to a wide incorporation obsessed with various everyday materials such as cosmetics or dress, optical devices, catalytic, bactericidal, electronics, sensor technology, biological labelling and treatment of some cancers. Nanoparticles can be chemically or biologically synthesized. This review focuses on the need to develop nanoparticles, advantages, disadvantages, synthesis, properties, applications of nanoparticles exist in different forms. Nanoparticles are very capable in selective tumour contact cancer therapy due to their small size and modifiability. These particles consist of pure active pharmaceutical ingredients and are stabilized regularly with surfactant. Nanoparticles are tiny materials that have particle sizes in the range of 1 to 1000 nm.
Bio-based nanotechnology has its existence in biological dimensions e.g. microbial cell factories (bacteria, fungi. algae, yeast, cyanobacteria) plants, and biopolymers. They provide multipurpose biological platforms to supply well-designed materials for diverse nano-biotechnological applications. The “green or bio-based synthesis of nanoparticles (NPs)” has witnessed a research outburst in the past decade. The bio-based synthesis of NPs using microbial cell factories is a benign process and requires mild conditions for the synthesis with end products being less/non-toxic. As a result, its application has extended in multitudinous industries including environment, cosmetics, and pharmaceutical. Thus, the present review summarizes all the significant aspects of nanotechnology and the reason to switch towards the bio-based synthesis of NPs using microbial cell factories. It consists of a detailed description of the bio-based methods employed for the synthesis and classification of NPs. Also, a comprehensive study on the application of bio-based NPs in the various industrial and biotechnological domains has been discussed. The limitation and its solution would help identify the applicability of NPs to “identified and unidentified” sectors.
Non-viral vehicles hold therapeutic promise in advancing the delivery of a variety of cargos in vitro and in vivo, including small molecule drugs, biologics, and especially nucleic acids. However, their efficacy at the cellular level is limited by several delivery barriers, with endolysosomal degradation being most significant. The entrapment of vehicles and their cargo in the acidified endosome prevents access to the cytosol, nucleus, and other subcellular compartments. Understanding the factors that contribute to uptake and intracellular trafficking, especially endosomal entrapment and release, is key to overcoming delivery obstacles within cells. In this review, we summarize and compare experimental techniques for assessing the extent of endosomal escape of a variety of non-viral vehicles and describe proposed escape mechanisms for different classes of lipid-, polymer-, and peptide-based delivery agents. Based on this evaluation, we present forward-looking strategies utilizing information gained from mechanistic studies to inform the rational design of efficient delivery vehicles.
Human exposure to nanoparticles has been dramatically increased in the past 25 years as a result of the rapidly developing field of nanotechnology. Many have recognized the importance of identifying potential effects on human health associated with the manufacture and use of these important technology. Many questions remain unanswered regarding the short- and long-term effect, systemic toxicity, and carcinogenicity. Engineered nanoparticles can be taken up by the human body via inhalation, ingestion, dermal uptake, and injection. They can reach the bloodstream and ultimately affect multiple body organs such as liver and spleen or even transcend the blood-brain barrier. Because of the huge diversity of materials used and the wide range in size of nanoparticles, these effects will vary a lot. Local and systemic adverse effects consist of primarily inflammatory reactions. Other observed effects include generation of reactive oxygen species and subsequent oxidative stress, disruption of proteins, DNA, mitochondria and membrane structures, as well as changes in cell signaling pathways.
Tuberculosis (TB) remains a major global threat and has the second-highest mortality rate of infectious diseases. The conventional pharmacotherapy of tuberculosis fails primarily due to inappropriate drug prescriptions, poor patient compliance, and the emergence of multidrug-resistant strains. The chronicity of multiple drug administration is also associated with several side effects and organ toxicity. There are many tools for TB detection such as chest X-rays, blood specimen cultures, and skin tests, but the preferred one is sputum smear microscopy. The above methods have their limitations in terms of cost, time constraints, low diagnostic sensitivity, etc. Medical science is therefore in utmost need of finding new sensitive nanodiagnostic tests that can be fast, inexpensive, and capable of processing any biological sample. Also, there is a need for a better therapeutic regimen involving lower dose frequencies, higher drug concentrations at the infected site, reduced drug-induced toxicities, higher bioavailability, and minimized chances of TB drug resistance. This chapter discusses the clinical perspectives of nanotechnology toward targeting MTB as well as presents a review of various types of nanoparticles for the detection of mycobacterial species in TB diagnosis and treatment. Nanoparticles in the form of nanosuspensions, niosomes, carbon nanotubes, quantum dots, nanoemulsions, and aerosol nanoparticles provide new opportunities for improving both diagnosis and antitubercular treatment.
For the past few decades, there has been a major investigate intrigues among the zone of medicate conveyance utilizing particulate conveyance frame works as carriers for the limited and expansive particles. Particulate frameworks like nanoparticles are utilizing as a physical approach to alter and move forward the pharmacological medicine and pharmacodynamics properties of various forms of sedate atoms. Nanoparticles area unit characterized as a particles with a breadth littler than 100nm, area unit increasingly utilize in many applications, count medicate framework and to pass organ obstructions like the blood brain barrier. Nano crystals and alternative nanoparticles are obtaining a parcel of thought for potential utilize in medicine, and medicine sedate revelation. The use of engineering science and prescription drugs and additional significantly sedate connivance is ready to unfold quickly. Right away various substances area unit at a lower place examination for sedate provides. Keywords: Nanoparticles, Types, Method of Preparation, Evaluation, Applications.
Mitochondrial malfunction plays a crucial role in cancer development and progression. Cancer cells show a substantially higher mitochondrial activity and greater mitochondrial transmembrane potential than normal cells. This concept can be exploited for targeting cytotoxic drugs to the mitochondria of cancer cells using mitochondrial-targeting compounds. In this study, a polyamidoamine dendrimer-based mitochondrial delivery system was prepared for curcumin using triphenylphosphonium ligands to improve the anticancer efficacy of the drug in vitro and in vivo. For the in vitro evaluations, various methods, such as viability assay, confocal microscopy, flow cytometry, reactive oxygen species (ROS), and real-time polymerase chain reaction analyses, were applied. Our findings showed that the targeted-dendrimeric curcumin (TDC) could successfully deliver and colocalize the drug to the mitochondria of the cancer cells, and selectively induce a potent apoptosis and cell cycle arrest at G2/M. Moreover, at a low curcumin dose of less than 25 μM, TDC significantly reduced adenosine triphosphate and glutathione, and increased the ROS level of the isolated rat hepatocyte mitochondria. The in vivo studies on the Hepa1-6 tumor-bearing mice also indicated a significant tumor suppression effect and the highest median survival days (Kaplan-Meier survival estimation and log-rank test) after treatment with the TDC construct compared to the free curcumin and untargeted construct. Besides its targeted nature and safety, the expected improved solubility and stability represent the prepared targeted-dendrimeric construct as an up-and-coming candidate for cancer treatment. The results of this study emphasize the promising route of mitochondrial targeting as a practical approach for cancer therapy, which can be achieved by optimizing the delivery method.
Human exposure to nanoparticles has been dramatically increased in the past 25 years as a result of the rapidly developing field of nanotechnology. Many have recognized the importance of identifying potential effects on human health associated with the manufacture and use of these important technology. Many questions remain unanswered regarding the short- and long-term effect, systemic toxicity, and carcinogenicity. Engineered nanoparticles can be taken up by the human body via inhalation, ingestion, dermal uptake, and injection. They can reach the bloodstream and ultimately affect multiple body organs such as liver and spleen or even transcend the blood-brain barrier. Because of the huge diversity of materials used and the wide range in size of nanoparticles, these effects will vary a lot. Local and systemic adverse effects consist of primarily inflammatory reactions. Other observed effects include generation of reactive oxygen species and subsequent oxidative stress, disruption of proteins, DNA, mitochondria and membrane structures, as well as changes in cell signaling pathways.
Building upon our previous studies on interactions of amphiphilic Janus nanoparticles with glass-supported lipid bilayers, we study here how these Janus nanoparticles perturb the structural integrity and induce shape instabilities of membranes of giant unilamellar vesicles (GUVs). We show that 100 nm amphiphilic Janus nanoparticles disrupt GUV membranes at a threshold particle concentration similar to that in supported lipid bilayers, but cause drastically different membrane deformations, including membrane wrinkling, protrusion, poration, and even collapse of entire vesicles. By combining experiments with molecular simulations, we reveal how Janus nanoparticles alter local membrane curvature and collectively compress the membrane to induce shape transformation of vesicles. Our study demonstrates that amphiphilic Janus nanoparticles disrupt vesicle membranes differently and more effectively than uniform amphiphilic particles.
Dendrimer synthesis and potential applications of the lysine-based dendrimers as gene carrier systems and nano-fluorescent probes have been explored. An intrinsically fluorescent polylysine dendrimer (DM1), (Gly)(Lys)63(NH2)64, was synthesized by both Fmoc- (30% overall yield) and Boc-SPPS (64% overall yield) by divergent method and characterized by NMR, MS and RP-HPLC. Boc-SPPS is a recommended method for synthesizing this compound which provided higher yield and purer product. Diffusibility of the DM1 in various types of media and in cells has revealed the difference between in vitro and in vivo transport. In concentrated and more complex media (20-60%(v/v) glycerol solutions, HPMC and actin gels) when beyond the limits of PCS, the FRAP technique allowed the diffusion coefficients (D) of DM1 to be accessed. For DNA delivery, DM1, DM2 ((C18)(Lys)7(NH2)8) and DM3 ((C18)(Lys)7(NH2)7(RGD)1) were capable of condensing DNA. The DNA was better condensed by DM2 and DM3 possibly due to the synergistic effects between the electrostatic and hydrophobic interactions. At charge ratios of 20:1 (DM1) and 10:1 (DM2 and DM3) (+/-) a 4h incubation period produced optimal transfection. Confocal microscopy and luciferase assay indicated transfection efficiencies of the dendrimers in descending order: DM3>DM2>LPP(Lipofectamine-Plus™)>DM1>pDNA. As a nano-fluorescent probe (DM1), the method for studying the dynamic uptake of this fluorescent probe has been established and allowed the D values to be calculated. Values were found in the range from 5.99x10-11 cm2 s-1 (in SK/MES-1 cells) to 9.82x10-11 cm2 s-1 (in Caco-2 cells) for the dendrimer and 2.36x10-11 cm2 s-1 (in Caco-2 cells) for the dendriplexes. The difference implied variation in the intracellular architecture of the cell types and the effect of particle size. Using the DM1 with other fluorescent dyes the excitation/emission spectra of the dyes and the concentration used of the DM1 must be considered to avoid the contribution of the DM 1 in emission spectrum of the dyes. The uptake of both dendrimer and dendriplexes entailed endocytosis which was reduced by the effect of medium flow. The DM1 dendrimer clearly interacted with actin filaments via the electrostatic interaction as confirmed by the TEM, SEM and SDS-PAGE results. Actin formed reversible insoluble complexes with DM1 indicating none of chemical bonding within the complexes reinforced by results from Western blots. DM1 exhibited biphasic effects on the actin polymerisation process depending on its concentration. It displayed inhibitor and activator behaviour, respectively, at low and high concentration. It was concluded that DM1, DM2 and DM3 serve as potential tools for gene delivery whereas their unique architectures still allow them to be engineered for indefinite applications in the future.
Skin offers an easily accessible site for drug administration as well as for health signal monitoring, with non‐invasiveness or minimal‐invasiveness, convenience, and good patient compliance. Transdermal theranostics promises for personalized, home‐based, and long‐term management of chronic diseases, and is expected to change the landscape of healthcare profoundly. In this article, we review the recent advances in transdermal drug delivery, diagnosis based on sweat and skin interstitial fluid, and wearable devices. The advantages, limitations, and commercialization of these emerging techniques are comparatively discussed. Skin offers an easily accessible site for drug administration as well as for health signal monitoring, with non‐noninvasiveness or minimal‐invasiveness, convenience, and good patient compliance. Transdermal theranostics promises for personalized, home‐based, long‐term management of chronic diseases.
Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM2-3Glu, significantly affect the type and extent of interaction with biological structures.
Background:
Understanding the structure of hybrid nanoparticle-lipid multilayers is of fundamental importance to their bioanalytical applications and nanotoxicity, where nanoparticle-membrane interactions play an important role. Poly(amidoamine) (PAMAM) dendrimers are branched polymeric nanoparticles with potential biomedical applications due to precise tunability of their physicochemical properties. Here, the effect of PAMAM dendrimers (2.9-4.5 nm) with either a hydrophilic amine (NH2) or a hydrophobic C12 chain surface termination on the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilayers has been studied for the first time.
Methods:
DOPC multilayers were created by the liposome-rupture method via drop-casting dendrimer-liposome dispersions with the dendrimers added at different concentrations and at three different stages. The multilayer structure was evaluated via the analysis of the synchrotron X-ray reflectivity (XRR) curves, obtaining the bilayer d-spacing, the coherence length from the Scherrer (Ls) analysis of the Bragg peaks, and the paracrystalline disorder parameter (g).
Results:
Dendrimer addition led to lipid bilayer thinning and more disordered multilayer structures. Larger hydrophobic dendrimers caused greater structural disruption to the multilayers compared to the smaller dendrimers. The smallest, positively charged dendrimers at its highest concentration caused the most pronounced bilayer thinning. The dendrimer-liposome mixing method also affected the multilayer structure due to different dendrimer aggregation involved.
Conclusions:
These results show the complexity of the effect of dendrimer physicochemical properties and the addition method of dendrimers on the structure of mixed dendrimer-DOPC multilayers.
General significance:
These insights are useful for fundamental understanding of nanotoxicity and future biomedical application of nanocomposite multilayer materials in which nanoparticles are added for enhanced properties and functionality.
Understanding interactions between nanoparticles and model membranes is relevant to functional nano-composites and the fundamentals of nanotoxicity. In this study, the effect of polyamidoamine (PAMAM) dendrimers as model nanoparticles (NP) on the mesophase behaviour of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) has been investigated using high-pressure small-angle X-ray scattering (HP-SAXS). The pressure-temperature (p−T) diagrams for POPE mesophases in excess water were obtained in the absence and presence of G2 and G4 polyamidoamine (PAMAM) dendrimers (29 Å and 45 Å in diameter, respectively) at varying NP-lipid number ratio (ν = 0.0002–0.02) over the pressure range p = 1–3000 bar and temperature range T = 20–80 °C. The p−T phase diagram of POPE exhibited the Lβ, Lα and HII phases. Complete analysis of the phase diagrams, including the relative area pervaded by different phases, phase transition temperatures (Tt) and pressures (pt), the lattice parameters (d-spacing), the pressure-dependence of d-spacing (Δd/Δp), and the structural ordering in the mesophase as gauged by the Scherrer coherence length (L) permitted insights into the size- and concentration-dependent interactions between the dendrimers and the model membrane system. The addition of dendrimers changed the phase transition pressure and temperature and resulted in the emergence of highly swollen lamellar phases, dubbed Lβ-den and Lα-den. G4 PAMAM dendrimers at the highest concentration ν = 0.02 suppressed the formation of the HII phase within the temperature range studied, whereas the addition of G2 PAMAM dendrimers at the same concentration promoted an extended mixed lamellar region in which Lα and Lβ phases coexisted.
Statement of Significance
Using high pressure small angle X-ray scattering in the pressure range 1–3000 bar and temperature range 20–60 °C, we have studied interactions between PAMAM dendrimers (as model nanoparticles) and POPE lipid mesophases (as model membranes). We report the pressure-temperature phase diagrams for the dendrimer-lipid mesophases for the first time. We find that the dendrimers alter the phase transition temperatures (Tt) and pressures (pt), the lattice parameters (d-spacing), and the structural order in the mesophase. We interpret these unprecedented results in terms of the fluidity of the lipid membranes and the interactions between the dendrimers and the membranes. Our findings are of fundamental relevance to the field of nanotoxicity and functional nanomaterials that integrate nanoparticles and organized lipid structures.
Microvesicles (MVs) are used by various types of cells in the human body for intercellular communication, making them biomarkers of great potential for the early and non-evasive diagnosis of a spectrum of diseases. An integrated analysis including morphological, quantitative, and compositional studies is most desirable for the clinical application of MV detection; however, such integration is limited by the currently available analysis techniques. In this context, exploiting the phosphatidylserine (PS) exposure of MVs, we synthesized a series of dendritic molecules with PS-binding sites at the periphery. PS-dendron binding was studied at the molecular level using NMR approaches, whereas PS-containing membrane-dendron interaction was investigated in an aqueous environment using plasmon waveguide resonance spectroscopy. As a proof of concept, polyethylene terephthalate surface was functionalized with the synthetic dendrons, forming devices that can capture MVs to facilitate their subsequent analyses.
To develop a full-biodegradable thermoresponsive polymer with a wide temperature range, a hydrophilic, biodegradable and biocompatible thermoresponsive polymer having a polylactide backbone polymer and a side chain norbornene group was synthesized by ring-opening polymerization of a norbornene-functionalized lactide, followed by graft of backbone polymer with polyethylene glycol monomethyl ether (MPEG) and modification with isopropylamide (IPA), the latter of which critically influences the phase transition temperature of the copolymer. The effects of polymerization degree (DP), molecular weight of MPEG, the proportion of MPEG and IPA, and the concentration of polymer on the behaviors of the thermoresponsive polymer solution were systematically studied. The Cp ranged from 32 to 72 °C, and it was found that higher number of the MPEG moiety led to higher Cp, whereas the longer isopropylamide group resulted in lower Cp. It is noteworthy that with the unique characteristics, thermosensitivity at body temperature, biodegradability, and a well-defined copolymer structure, the developed thermoresponsive polymer is promising for biomedical applications as an essential material.
A systematic evaluation of the accuracy of the GROMOS-compatible 2016H66 forcefield in the simulation of dendrimers is performed. More specifically, the poly(amido amine) (PAMAM) and the poly(propylene imine) (PPI) are considered due to the availability of experimental data and simulation results in the literature. A total of 36 molecular systems are simulated and the radius of gyration, asphericity, density profiles and the self-diffusion coefficients are monitored in terms of the generation number and pH (low, medium and high) condition. Overall, the results support the recommendation of the 2016H66 forcefield for the simulation of dendrimer systems. The natural building-block based strategy adopted in the definition of 2016H66, together with a careful parametrization of the chemical functional groups to reproduce thermodynamic properties in environments of different polarity, and also the ability to accurately reproduce the expected structural and dynamic features of dendrimers, as shown in the present work, make this forcefield an attractive option for the simulation of such systems and alike.
The widespread use of antibiotics has led to an increase in the number of strains resistant to major antibacterial pharmaceuticals. Many synthesized quaternary ammonium compounds possess antibacterial, antifungal and antiviral properties. Incorporation of quaternary ammonium moieties into dendrimers represents a promising strategy for the preparation of novel antimicrobial biomaterials. Here, poly(quaternary ammonium) chloride dendrimers were synthesized by functionalizing poly(propylene imine) (PPI) dendrimer with various lengths of alkyl chains. In vitro antimicrobial assays indicate that the amphiphilic dendrimers are potent antimicrobial agents with activity against multidrug-resistant pathogens such as the methicillin-resistant Staphylococcus aureus.
L'utilisation de nanoparticules (NPs) comme vecteur de médicaments est actuellement en plein développement de vectoriser des principes actifs dans l'organisme pour en diminuer les effets secondaires. Cependant peu d'études portent sur l'activité biologique de ces NPs et plus particulièrement sur leur toxicité intrinsèque. Récemment, une formulation orale d'Héparine de Bas Poids Moléculaire à base de NPs composées d'un mélange de deux polymères, polycaprolactone et Eudragit® RS, a été développée au sein de l'EA3452. Nous avons entrepris d'étudier la cytotoxicité de ces NPs vides ainsi que celles chargées avec le médicament par deux méthodes d'encapsulation : la nanopréciptation et la double émulsion. La toxicité observée des NP vides a été étudiée. Elles sont toxiques pour la lignée de macrophages de rat NR8383 d'une manière dose- et temps-dépendante. La microscopie électronique a montré que les NPs pénétraient dans la cellule, de façon unitaire, par endocytose et gagnent les mitochondries déclenchant un phénomène de mitophagie alors qu'aucune image évoquant une apoptose n'a été observée. Ceci est confirmé par l'étude des voies métaboliques par « microchip arrays » et RT-PCR quantitative. Les voies métaboliques conduisant à l'autophagie sont activées (en particulier le gène atg1611) sans que l'apoptose soit mise en jeu. La désorganisation des structures mitochondriales est associée à une répression de l'expression du gène opa1. Ces résultats obtenus doivent être confirmé sur des macrophages humains. Cependant ils tendent à montrer que les NPs seules ont des effets biologiques qui doivent être pris en compte avant toute utilisation chez l'homme
This paper describes the first synthesis of a new class of topological macromolecules which we refer to as “starburst polymers.” The fundamental building blocks to this new polymer class are referred to as “dendrimers.” These dendrimers differ from classical monomers/oligomers by their extraordinary symmetry, high branching and maximized (telechelic) terminal functionality density. The dendrimers possess “reactive end groups” which allow (a) controlled moelcular weight building (monodispersity), (b) controlled branching (topology), and (c) versatility in design and modification of the terminal end groups. Dendrimer synthesis is accomplished by a variety of strategies involving “time sequenced propagation” techniques. The resulting dendrimers grow in a geometrically progressive fashion as shown: Chemically bridging these dendrimers leads to the new class of macromolecules—”starburst polymers” (e.g., (A)n, (B)n, or (C)n).
Starburst dendrimers are novel, water-soluble polymeric materials, with a well-defined composition and structure. In our application, we used dendrimers composed of poly(amidoamine) groups to which we coupled several specific antibodies, to investigate potential formats based on radial partition immunoassay. The coupled antibodies have retained their stability and immunological binding after coupling, both in solution and when immobilized onto a solid support. On the basis of our feasibility studies with model systems, we conclude that immunoassays can be developed with performance equivalent to or better than that in many established systems. By application of a mixture of the dendrimer-coupled antibody and the analyte of interest to the solid phase, we have investigated the performance characteristics of solution-phase immunoassays. Our experiments demonstrate enhanced sensitivity for creatine kinase MB isoenzyme (CKMB), thyrotropin, and myoglobin assays and reduced instrumental analysis time for the CKMB assay.
Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.
Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.
This study demonstrates in vivo effectiveness of a nonviral vector system, Epstein-Barr virus (EBV)-based plasmid vector coupled with polyamidoamine (PAMAM) dendrimer (EBV/polyplex), in suicide gene therapy of cancer. The EBV-based vector is a plasmid vector containing EBV nuclear antigen 1 (EBNA1) gene and oriP from EBV genome. HSV-1 tk gene was transferred into Ewing's sarcoma cell lines, A4573 and KP-EWS-YI, by using an EBV-based plasmid vector, pSES.Tk, or a conventional plasmid vector, pS.Tk. Cells transfected with pSES.Tk/dendrimer showed approximately 100 times lower ID50 to ganciclovir (GCV) compared with those transfected with pS. Tk/dendrimer. Intratumoral injection of pSES.Tk/dendrimer but not pS. Tk/dendrimer drastically suppressed the growth of tumors which had generated from A4573 or Huh7 hepatocellular carcinoma (HCC) cells inoculated into severe combined immunodeficiency (SCID) mice. The treatment with pSES.Tk/dendrimer also resulted in significant prolongation of survival of the mice implanted with A4573. These results suggest that the EBV/polyplex system could be useful for in vivo suicide gene therapy of cancer. Gene Therapy (2000) 7, 53-60.
We sought to develop nanoscale drug delivery material that would allow targeted intracellular delivery while having an imaging capability for tracking uptake of the material. A complex nanodevice was designed and synthesized that targets tumor cell through the folate receptor.
The device is based on an ethylenediamine core polyamidoamine dendrimer of generation 5. Folic acid, fluorescein, and methotrexate were covalently attached to the surface to provide targeting, imaging, and intracellular drug delivery capabilities. Molecular modeling determined the optimal dendrimer surface modification for the function of the device and suggested a surface modification that improved targeting.
Three nanodevices were synthesized. Experimental targeting data in KB cells confirmed the modeling predictions of specific and highly selective binding. Targeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug.
These results demonstrate the ability to design and produce polymer-based nanodevices for the intracellular targeting of drugs, imaging agents, and other materials.
Experimental data indicates that poly(amidoamine) (PAMAM) dendrimers flatten when in contact with a substrate, i.e. they are no longer spherical, but resemble flat disks. In order to better understand the deformation behavior of these branched polymers, a series of atomistic molecular dynamics simulations is performed. The resulting flattened dendrimer conformations are compared to atomic force microscopy (AFM) images of individual dendrimers at air/mica and water/mica interfaces. The ability of the polymers to deform is investigated as a function of dendrimer generation (2-5) and the required energies are calculated. Our modeling results show good agreement with the experimental AFM images, namely that dendrimers are highly flexible and capable of forming multiple interaction sites between most of their branch ends and the substrate. The deformation energy scales with dendrimer generation and does not indicate an increase in stiffness between generations 2 and 5 due to steric effects.
Starburst polyamidoamine (PAMAM) dendrimers are a new synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and antisense expression plasmids for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA/dendrimer complexes to transfer oligonucleotides and plasmid DMA to mediate antisense inhibition was assessed in in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonuc-leotides or antisense cDNA plasmids using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25 - 50% reduction of baseline luciferase activity, depending on the oligonucleotide or antisense plasmid DNA concentrations, generation of the dendrimer and charge ratio of the DNA/dendrimer complexes. Binding of the oligonucleotide to the dendrimer extended intracellular survival of the unmodified DNA. Complexing with dendrimers does not change sequence specificity of the antisense oligonucleotides or RNA. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.
Starburst dendrimers (SBDs) of different generations (size) and level of protonations of their surface amino groups and solutions of dimyristoylphosphatidylcholine (DMPC) vesicles were analyzed, both separately and in mixtures of the two components, by negative-staining transmission electron microscopy (TEM), dynamic light scattering (DLS), and, mainly, by computer-aided continuous wave (CW)- and pulsed-electron paramagnetic resonance (EPR). For the EPR study, the SBDs were labeled with a nitroxide radical (SBD-T). TE micrographs showed the vesicles as multilamellar structures of spherical shape with diameters ranging from 0.2 to 1.2 m. DLS measurements provided the mean vesicle diameter (d) at ca. 400 nm, whereas the diameter of generation 6 was 7 nm. No large-sized permanent supramolecular structures (d > 400 nm) were formed. EPR measurements at room temperature were poorly informative, since (1) a fraction of the dendrimers was not interacting with the vesicles, and (b) the labels that were interacting with the vesicles were rotating quickly around the main axis. Interactions between the dendrimers and the vesicles (tested by a decrease in rotational mobility of the label) became EPR-observable and quantifiable below the freezing transition of a portion of the solution, which could not be detected by EPR analysis. The fraction of the dendrimers interacting with the vesicles underwent a glass transition. Dendrimer−vesicle interactions modified the direction of the fast-rotation axis of the radical, and the interaction was more effective for protonated dendrimers of a larger size, i.e., later generation. A “complex” was formed between one solvent molecule and the nitroxide radical. Interactions between the SBD-T and the vesicle partially compressed the hydration layer of the N−O group, and/or the hydration layer of the vesicle headgroups was compressed onto the unpaired-electron site. This study provides information on the possible utilization of starburst dendrimers as gene carriers.
IN THE CONTEXT OF DRUG DELIVERY, THE NEEDS FOR MATErials can generally be broken into two categories, notes Robert S. Langer, a professor of chemical and biomedical engineering at Massachusetts Institute of Technology: the creation of new materials and better understanding of how to manipulate existing materials.
Polyamidoamine dendrimers have been shown to be very effective at transporting DNA across cell membranes in transfection experiments. To investigate the membrane interactions with dendrimers that could contribute to this efficacy, the ability of dendrimers to permeabilize lipid vesicles in suspension has been studied. Vesicles were prepared containing the self-quenching, membrane impermeant dye calcein, and were treated with dendrimers of different sizes. Increase in fluorescence was attributed to release of the dye. Membranes containing dioleoyl phosphatidylethanolamine and stearic or oleic acid, lipids with a preference for non-lamellar phases, were very susceptible to disruption by dendrimers, with larger dendrimers being more effective than smaller. However, membranes containing lipids with a preference for the lamellar phase (either pure phosphatidylcholine, or phosphatidylcholine:phosphatidylserine) were largely unaffected. The concentration dependence of the permeabilization strongly suggests an aggregation-mediated mechanism for membrane disruption. Requenching measurements using cobalt citrate showed that permeabilization did not occur uniformly among the vesicles, but rather was all-or-none, with a subpopulation of vesicles responsible for essentially all of the dye release. This is also suggestive of an aggregation-induced response. Lastly, although osmotic forces are thought to play an important role in dendrimer-mediated transfection, we observed no effect of osmotic pressure and membrane tension on the efficacy of dendrimers in solution. It is likely that, in cells, dendrimers traverse cell membranes via endosomes, and the entrapment of the dendrimer itself within the endosomal vesicle may be a key factor in its ability to cause rupture.
Polyamidoamine dendrimers were modified chemically by reaction with DOTA and DTPA type bifunctional metal chelators and were coupled to monoclonal antibody 2E4 without loss of protein immunoreactivity. Both the DTPA- and DOTA-dendrimer-antibody constructs were easily labeled with 90Y, 111In, 212Bi or cold Gd(III) suggesting use of this dendrimer-macrocycle for mAb guided radiotherapy or imaging.
Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.
Starburst dendrimers are three-dimensional, highly ordered oligomeric and polymeric compounds formed by reiterative reaction sequences starting from smaller molecules—“initiator cores” such as ammonia or pentaerythritol. Protecting group strategies are crucial in these syntheses, which proceed via discrete “Aufbau” stages referred to as generations. Critical molecular design parameters (CMDPs) such as size, shape, and surface chemistry may be controlled by the reactions and synthetic building blocks used. Starburst dendrimers can mimic certain properties of micelles and liposomes and even those of biomolecules and the still more complicated, but highly organized, building blocks of biological systems. Numerous applications of these compounds are conceivable, particularly in mimicking the functions of large biomolecules as drug carriers and immunogens. This new branch of “supramolecular chemistry” should spark new developments in both organic and macromolecular chemistry.
Individual dendrimer molecules of poly(amidoamine) (PAMAM) from generation 10 (G10) to G5 were imaged by conventional transmission electron microscopy (TEM) after staining with aqueous sodium phosphotungstate. The dendrimers were resolved as separate, beam-stable entities, and their sizes and distribution of sizes were statistically analyzed and compared with data from small-angle X-ray scattering in solution. Aqueous G10 dendrimers were also characterized by examination in the frozen, hydrated state after quench-freezing in liquid ethane (cryo-TEM) and the data compared with the results of the staining experiments. To a first approximation, the dendrimers appeared circular in projection and the diameters conformed to a Gaussian distribution which broadened somewhat with increasing generation number. Cryo-TEM, in general, confirmed the staining experiments but suggested that, in the native state, there is more variability in the shapes of the dendrimers, with polyhedral shapes occurring quite frequently.
This report describes a computer-aided CW- and pulsed-electron paramagnetic resonance (EPR) investigation on the structural modification of dimyristoylphosphatidylcoline (DMPC) vesicles, which occur upon interaction with starburst dendrimers (SBDs). Probes used for this study included doxyl-functionalized stearic acids, with the doxyl group attached at different positions of the stearic chain (5DXSA, 12DXSA, and 16DXSA). Mainly mobility and polarity parameters were evaluated from the analysis of the CW-EPR spectra, whereas the analysis of the decay and modulation of the electron spin−echo (ESE) signal provided information on the structural environment of the paramagnetic center. Due to the interaction with the SBD surface, the vesicle structure became more rigid and ordered. The enhanced rigidity of the structure also caused the tilting of the chains of about 50° with respect to the surface line. The permeability of water at the chain beginning level increased, thus increasing the rotational mobility of the probe. The perturbing effects lessened toward the end of the chains. A fraction of 16DXSA (15%) was in the bent conformation, with the chain inserted into the lipid layer and the two polar heads at the external surface. The interaction with protonated dendrimers caused the swelling of the vesicle structure. This study indicated that the bilayer structure is modified but only partially perturbed by the addition of the dendrimers, and the integrity of the vesicle, as a model cell membrane, is preserved after the interaction with the dendrimers. This is encouraging for the use of the SBDs as drug and gene carriers.
Carbon nanoprobes were utilized in tapping mode atomic force microscopy investigations to highlight the topographic differences between poly(amidoamine) (PAMAM) dendrimers, two-dimensional arrays of PAMAM dendrimers, and PAMAM core−shell tecto(dendrimers). The PAMAM core−shell tecto(dendrimers) used for this study consist of a shell of generation five (G5) dendrimers covalently linked to a G7 core. The volumes measured for the PAMAM core−shell tecto(dendrimers) suggest a 75−100% saturation of shell G5 dendrimers about the G7 core. This compares favorably with theoretical predictions that 12 G5 dendrimers should pack to form a shell about a G7 core. The effect of different imaging substrates was also explored. Tecto(dendrimers) imaged on hydrophobic surfaces experience a 6-fold increase in maximum peak height and a 3-fold decrease in diameter as compared to those imaged on mica. Measured volume is invariant.
The interactions between polyamidoamine starburst dendrimers (SBDs) and vesicles consisting of dimyristoylphosphatidylcholine (DMPC) and its phosphatidylcholate salt (DMPA-Na) in various relative ratios (from 0% to 40% of DMPA-Na) were studied by electron paramagnetic resonance (EPR) and by transmission electron microscopy (TEM). The EPR spectra were computer-simulated to extract mobility and structural parameters of the probes and labels. The systems were analyzed as a function of the level of protonation of SBDs and generation (G = 2SBD and G = 6SBD). Both the hydrophobic 5doxylstearic spin probe (5DSA) and the positively charged nitroxide CAT16, a cationic surfactant with a C16 chain, inserted in the vesicles and monitored the formation of dendrimer−mixed vesicle (DMPC/DMPA-Na) complexes. 5DSA revealed a partial ordering of a double-layer-like structure occurring at the dendrimer surface. The spin labels at the vesicle/dendrimer interface modified the structural and mobility parameters upon complexation with the dendrimer. At high levels of protonation, dendrimers showed a larger interaction with the vesicles, especially for the mixed vesicles, compared to dendrimers at a lower level of protonation. In agreement with EPR results, TEM micrographs showed that the addition of DMPA-Na to DMPC modifies the vesicle shape from spherical to rodlike. The EPR analysis suggested that the vesicles wrap around the large dendrimers whereas the small dendrimer directly interacts with the vesicle surface. The results are very promising for a better understanding of the mechanism of interaction of dendrimers with cell membranes which may be an important feature in the role of SBDs as drug and gene carriers to target cells.
Preformed poly(amidoamine) (PAMAM) dendrimer modules were self-assembled by using charge neutralization as the organizing force to first form supramolecular core-shell dendrimer assemblies. Subsequent introduction of a carbodiimide reagent was then used to produce in-situ amide bond formation at the amine-carboxylic acid contact points residing at the [dendrimer-core]-(dendrimer shell) interface.
PAMAM Starburst™ dendrimers are spherical macromolecules composed of repeating polyamidoamino units. They can be produced in successive “generations,” each with a defined size, molecular weight, and number of terminal amino groups. Because of these well-defined characteristics, PAMAMs are finding utility in a variety of applications, many of which are biological in nature. Little is known, however, about the biological behavior of the PAMAMs, which is critical to their use in vivo. Generation 3 (G3; MW = 5,147; 24 terminal amines), 5 (G5; MW = 21,563; 96 amines), and 7 (G7; MW = 87,227; 384 amines) PAMAMs were studied in V79 cells or in Swiss-Webster, mice for a number of biological properties, including (1) in vitro toxicity, (2) in vivo toxicity, (3) immunogenicity, and (4) biodistribution. Potential biological complications were observed only with G7 at the highest level tested. No evidence of immunogenicity was seen. The biodistribution properties of the Starburst™ dendrimers were rather unusual. G3 showed the highest accumulation in kidney tissue (∼15% ID/g over 48 h); G5 and G7 appeared to preferentially localize in the pancreas (peak levels ∼32% ID/g at 24 h, and ∼20% ID/g at 2 h, respectively). In addition, G7 showed extremely high urinary excretion, with values of 46 and 74% ID/g at 2 and 4 h, respectively. In general, the dendrimers did not exhibit properties that would preclude their use in biological applications. Depending on the situation (desired endpoint, dose, and generation used), however, the biodistribution of biological preparations should be carefully studied. © 1996 John Wiley & Sons, Inc.
Dendrimers are highly branched macromolecules of low polydispersity that provide many exciting opportunities for design of novel drug-carriers, gene delivery systems and imaging agents. They hold promise in tissue targeting applications, controlled drug release and moreover, their interesting nanoscopic architecture might allow easier passage across biological barriers by transcytosis. However, from the vast array of structures currently emerging from synthetic chemistry it is essential to design molecules that have real potential for in vivo biological use. Here, polyamidoamine (PAMAM, Starburst™), poly(propyleneimine) with either diaminobutane or diaminoethane as core, and poly(ethylene oxide) (PEO) grafted carbosilane (CSi–PEO) dendrimers were used to study systematically the effect of dendrimer generation and surface functionality on biological properties in vitro. Generally, dendrimers bearing -NH2 termini displayed concentration- and in the case of PAMAM dendrimers generation-dependent haemolysis, and changes in red cell morphology were observed after 1 h even at low concentrations (10 μg/ml). At concentrations below 1 mg/ml CSi–PEO dendrimers and those dendrimers with carboxylate (COONa) terminal groups were neither haemolytic nor cytotoxic towards a panel of cell lines in vitro. In general, cationic dendrimers were cytotoxic (72 h incubation), displaying IC50 values=50–300 μg/ml dependent on dendrimer-type, cell-type and generation. Preliminary studies with polyether dendrimers prepared by the convergent route showed that dendrimers with carboxylate and malonate surfaces were not haemolytic at 1 h, but after 24 h, unlike anionic PAMAM dendrimers they were lytic. Cationic 125I-labelled PAMAM dendrimers (gen 3 and 4) administered intravenously (i.v.) to Wistar rats (∼10 μg/ml) were cleared rapidly from the circulation (<2% recovered dose in blood at 1 h). Anionic PAMAM dendrimers (gen 2.5, 3.5 and 5.5) showed longer circulation times (∼20–40% recovered dose in blood at 1 h) with generation-dependent clearance rates; lower generations circulated longer. For both anionic and cationic species blood levels at 1 h correlated with the extent of liver capture observed (30–90% recovered dose at 1 h). 125I-Labelled PAMAM dendrimers injected intraperitoneally were transferred to the bloodstream within an hour and their subsequent biodistribution mirrored that seen following i.v. injection. Inherent toxicity would suggest it unlikely that higher generation cationic dendrimers will be suitable for parenteral administration, especially if they are to be used at a high dose. In addition it is clear that dendrimer structure must also be carefully tailored to avoid rapid hepatic uptake if targeting elsewhere (e.g. tumour targeting) is a primary objective.
It is suggested that Helfrich's concept of edge energy of a lipid bilayer explains the stability of black membranes and the ability of erythrocytes to reseal following hemolysis.
Poly(propylene imine) dendrimers having four or eight primary amino groups and a Starburst (PAMAM) dendrimer having eight primary amino groups were used as core molecules, to which phenylisothiocyanate derivatized (PITC) galbeta1-3galNAcbeta1-4[sialic acid alpha2-3]-galbeta1-4glc (oligo-GM1) residues were covalently attached to yield multivalent oligosaccharides. The synthesis of the oligo-GM1-PITC derivatized dendrimers was monitored using high performance thin layer chromatography, infrared spectroscopy, sialic acid content, and mass spectroscopy. The ability of multivalent oligo-GM1-PITC dendrimers to inhibit the binding of 125I-labeled cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1-coated microtiter wells was determined. IC50s obtained for the oligo-GM1-PITC dendrimers, GM1, and the oligosaccharide moiety of GM1 indicated that the derivatized dendrimers inhibited binding of the choleragenoid and the heat labile enterotoxin to GM1-coated wells at a molar concentration five- to 15-fold lower than native GM1 and more than 1,000-fold lower than that of the free oligosaccharide.
Using specular reflection of neutrons, we investigate for the first time the structure of a single dimyristoylphosphatidylcholine bilayer adsorbed to a planar quartz surface in an aqueous environment. We demonstrate that the bilayer is strongly adsorbed to the quartz surface and is stable to phase state changes as well as exchange of the bulk aqueous phase. Our results show that the main phase transition is between the L alpha phase and the metastable L beta'* phase, with formation of the P beta' ripple phase prevented by lateral stress on the adsorbed bilayer. By performing contrast variation experiments, we are able to elucidate substantial detail in the interfacial structure. We measure a bilayer thickness of 43.0 +/- 1.5 A in the L alpha phase (T = 31 degrees C) and 46.0 +/- 1.5 A in the L beta'* phase (T = 20 degrees C). The polar head group is 8.0 +/- 1.5 A thick in the L alpha phase. The water layer between the quartz and bilayer is 30 +/- 10 A for the lipid in both the L alpha and L'* phase. Our results agree well with those previously reported from experiments using lipid vesicles and monolayers, thus establishing the feasibility of our experimental methods.
A method is described for the separation of ihosphatidylcholine vesicles, formed by prolonged altrasonic irradiation under nitrogen in 0.1 M buffered NaCl solution, by molecular sieve chromatography on large-pore agarose gels. One fraction of the separated vesicles was found to be homogeneous by the criteria of gel filtration, sedimentation velocity ultracentrifuoation, and electron microscopy. Experimental data which support the homogeneous vesicle as a shelllike structure, about 250 A in diameter, with a continuous phosphatidylcholine bilayer surrounding a volume of solvent are presented. The following physical parameters were established for the homogeneous vesicles: S20,w, 2.1 S; D20,w, 1.87 × 10-7 cm2 sec-1; ν̄, 0.9885 ml g-1; [η], 0.041 dl g-1; and vesicle weight, 2.1 × 106.
Phospholipid vesicles containing the transmembrane protein H-2Kk spontaneously fuse to form planar membranes when incubated on treated glass surfaces. Pattern photobleaching of fluorescent lipid probes indicates that these planar membranes are continuous and that the lipids are as mobile as they are in conventional fluid bilayers or monolayers. H-2Kk molecules in these planar membranes are immobile. These membranes stimulate cytotoxic T lymphocytes when cultured with immune spleen cells. The response to H-2Kk in planar membranes is greatly enhanced by the addition of supernatant from concanavalin A-stimulated spleen cells, indicating that relatively little antigen processing or presentation by accessory cells occurs. Cytotoxic T cells induced by purified alloantigen are found to be as susceptible to antibody blockade as are effectors from conventional mixed lymphocyte culture, where the antibody is directed against a T-cell surface antigen reputed to strengthen target cell adhesion through an interaction independent of major histocompatibility antigens.
Upon prolonged incubation at low pH, the turbidity of a dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicle (SUV) suspension underwent an initial increase followed by a decrease to below the starting value. Electron microscopic observation of the vesicle suspension, after the turbidity minimum was reached, revealed the formation of discoidal particles which were smaller than the original vesicles. NMR studies indicated that all the choline groups in the discoids are accessible to added chemical shift agent, corroborating the fragmentation of the vesicles. Formation of the discoids from DMPC multilamellar vesicle (MLV) was also observed but without going through the initial increase in turbidity. The DMPC was subsequently found to be hydrolyzed into lysophosphatidylcholine (lysoPC) and myristic acid under the same conditions that caused the fragmentation. That the discoidal fragment consisted of DMPC and its hydrolysis products, lysoPC and myristic acid, was verified by electron microscopic observation of discoids when these components were mixed at neutral pH. A 50% cholesterol content prevented the fragmentation of DMPC vesicles. A model for this discoidal complex, in which a patch of DMPC bilayer is surrounded at its periphery by lysoPC and myristic acid, is proposed.
Boron neutron capture therapy (BNCT) is based on the nuclear capture reaction that occurs when boron-10, a stable isotope, is irradiated with low-energy or thermal neutrons (< or = 0.025 eV) to yield high LET alpha particles and recoiling 7Li nuclei [10B + nth-->[11B]-->4He(alpha) + 7Li + 2.39 MeV]. Approximately 10(9) boron-10 atoms must be delivered to each target cell in order to sustain a lethal 10B(n,alpha)7Li reaction. If MoAbs are to be used for targeting boron-10, then it is essential that they recognize a surface membrane epitope that is highly expressed on tumor cells and that a large number of boron-10 atoms be attached to each antibody molecule. In order to heavily boronate MoAbs, we have utilized starburst dendrimers (SD), which are precise, spherical macromolecules composed of repetitive poly(amidoamino) groups. Second- and fourth-generation dendrimers, having 12 and 48 reactive terminal amino groups and molecular weights of 2414 and 10,632 Da, respectively, were boronated using an isocyanato polyhedral borane, Na(CH3)3NB10H8NCO. The boronated starburst dendrimers (BSD), in turn, were derivatized with m-maleimidobenzoyl N-hydroxysulfosuccinimide ester (sulfo-MBS). The MoAbIB16-6, which is directed against the murine B16 melanoma, was derivatized with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The MBS-derivatized BSD and SPDP-derivatized MoAb were reacted to yield stable immunoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)
Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 6/1 (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of 10% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at 150 micrograms dendrimer/mL in the absence of DNA and about 75% in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to 1:1. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the 1:1 complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pKa's (3.9 and 6.9) of the amines in the polymer. The low pKa's permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pKa's, and low toxicity make the dendrimers suitable for gene-transfer vehicles.
The authors developed a new method for delivering contrast agents to tumors and tumor cells. Gadolinium complexes of folate-conjugated dendrimer-chelates increased the longitudinal relaxation rate of tumor cells expressing the high-affinity folate receptor, hFR. The coupling of folate to polymeric chelates, composed of a dendrimer backbone, targets these chelates to endogenous folate binding proteins. These proteins exist in both the serum of patients with cancer and on the cell surface of many human cancers of epithelial origin.
The authors attached folic acid to a generation four ammonia core polyamidoamine dendrimer. The folate-dendrimer was reacted with 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid to form the polymeric chelate f-PAMAM-TU-DTPA. For fluorescent studies, the generation four dendrimer was reacted with fluorescein-5-isothiocyanate and carboxytetramethylrhodamine succinimidyl ester, followed by capping the remaining amines with succinic anhydride.
The study results show that cells accumulate the folate-conjugated dendrimer in a receptor specific manner. Tumor cells expressing the high-affinity folate receptor showed a 650% increase in the mean fluorescence. This increase occurred with a rapid rise to 325%, followed by a slow increase to 650%. It required both the expression of the hFR and the coupling of folic acid to the dendrimer. Excess free folic acid inhibited the binding of the folate conjugated polymer. Fluorescent microscopic study showed that the folate-conjugated dendrimer binds to the cell surface and is accumulated within the cells. Treatment of tumor cells that express the hFR with gadolinium complexes of the folate-conjugated polymeric chelate increases the longitudinal relaxation rate by 110%. This increase was inhibited by an excess of free folic acid.
These data demonstrate that folate-conjugated magnetic resonance imaging contrast agents represent a promising new approach to tumor targeting.
When a pharmaceutical agent is encapsulated within, or attached to, a polymer or lipid, drug safety and efficacy can be greatly improved and new therapies are possible. This has provided the impetus for active study of the design of degradable materials, intelligent delivery systems and approaches for delivery through different portals in the body.
Over the last decade, research in somatic gene therapy has focused on selected approaches to deliver therapeutic genes to cells both ex vivo and in vivo. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, nonviral gene medicines are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Nonviral technologies consist of plasmid-based expression systems containing a gene encoding a therapeutic protein and synthetic gene delivery systems. In addition to the therapeutic gene, plasmid-based expression systems contain other genetic sequences to control the in vivo production and secretion of a protein. They may include elements that prolong extrachromosomal gene expression, cell-specific promoters and, optionally, gene switches for enabling drug-regulated gene therapy. Unique gene delivery systems will be required depending upon the biology and (patho)physiology of the target tissue. This review provides a critical view of gene therapy with a major focus on advanced nonviral technologies to control the in vivo location and function of administered genes.
A polyamidoamine (PAMAM) dendrimer generation 3.5 with a sodium carboxylate surface was conjugated to cisplatin giving a dendrimer-platinate (dendrimer-Pt; 20-25 wt% platinum) which was highly water soluble and released platinum slowly in vitro. In vivo the dendrimer-Pt and cisplatin were equi-active i.p. against i.p. L1210, and at high dose dendrimer-Pt given i.p. showed activity against i.p. B16F10 whereas cisplatin did not. Additionally, when administered i.v. to treat a palpable s.c. B16F10 melanoma, the dendrimer-Pt displayed antitumor activity whereas cisplatin was inactive. Measurement of platinum levels in blood and tissues after i.v. injection of cisplatin (1 mg/kg) or dendrimer-Pt (15 mg/kg)-the maximum tolerated dose (MTD) of these compounds-showed selective accumulation of the dendrimer-Pt in solid tumor tissue by the EPR effect (a 50-fold increase in area under curve compared with cisplatin). The dendrimer-Pt was also less toxic (3- to 15-fold) than cisplatin and thus has potential for further investigation as a novel antitumor approach.
The membrane disruption properties of high generation (G4 to G7) poly(amidoamine) (PAMAM) dendrimers are evaluated and compared to linear poly(lysine). The G6 and G7 dendrimers are unusually effective at inducing leaky fusion of anionic, large unilamellar vesicles, as determined by standard fluorescence assays for lipid mixing, leakage, and contents mixing. Both G7 dendrimer and poly(lysine) are able to disrupt sterically stabilized vesicles that are coated with poly(ethylene glycol). A G7 dendrimer/DNA complex with a 1:1 concentration ratio of dendrimer surface amines to DNA phosphate groups is unable to induce leakage of 3:7 POPA-PE vesicles; however, extensive leakage is observed when the surface amine to phosphate stoichiometry is >/=3:1. Thus, the DNA/dendrimer complexes that typically induce high levels of cell transfection are also able to induce high levels of vesicle leakage. The G7 dendrimer does not induce membrane phase separation in 3:7 POPA-PE vesicles, but an inverse hexagonal phase is observed by (31)P NMR. The enhanced membrane disruption is interpreted in terms of a membrane bending model. A rigid, polycationic dendrimer sphere uses electrostatic forces to bend a malleable, anionic membrane and induce bilayer packing stresses. This bending model is biomimetic in the sense that protein-induced membrane bending is currently thought to be an important factor in the fusion mechanism of influenza virus.
The objective of this study was to determine the permeability of a series of poly amidoamine (PAMAM) dendrimers of generations 0-4 (G0-G4) across MDCK (Madin-Darby Canine Kidney) cell line. PAMAM dendrimers with incremental increase in size and molecular weight were labeled by fluorescein isothiocyanate (FITC) and the least polydisperse fractions were collected by size exclusion chromatography. MDCK cells were grown on Transwell filters for four days. The conjugates were detected by HPLC equipped with fluorescence detector. The permeability of the dendrimers across MDCK cells was determined in the apical to basolateral direction. The rank-order permeability of the PAMAM dendrimers was G4 > G1 approximately G0 > G3 > G2. The permeability of mannitol in the presence of G4 increased by nine-fold. Results suggest that the transepithelial transport of PAMAM dendrimers is effected by both the polymer size, and the modulation of the cell membrane by the cationic dendrimers.
Uniaxially aligned phospholipid bilayers are often used as model membranes to obtain structural details of membrane-associated molecules, such as peptides, proteins, drugs, and cholesterol. Well-aligned bilayer samples can be difficult to prepare and no universal procedure has been reported that orients all combinations of membrane-embedded components. In this study, a new method for producing mechanically aligned phospholipid bilayer samples using naphthalene, a sublimable solid, was developed. Using (31)P-NMR spectroscopy, comparison of a conventional method of preparing mechanically aligned samples with the new naphthalene procedure found that the use of naphthalene significantly enhanced the alignment of 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine to 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The utility of the naphthalene procedure is also demonstrated on bilayers of many different compositions, including bilayers containing peptides such as pardaxin and gramicidin. These results show that the naphthalene procedure is a generally applicable method for producing mechanically aligned samples for use in NMR spectroscopy. The increase in bilayer alignment implies that this procedure will improve the sensitivity of solid-state NMR experiments, in particular those techniques that detect low-sensitivity nuclei, such as 15N and 13C.
Lipids are integral components of all biological membranes. Understanding the physical and chemical properties of these lipids is critical to our understanding of membrane functions. We developed a new atomic force microscope (AFM) approach to visualize in real time the temperature-induced lipid phase transition and domain separation processes in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes and estimate the thermodynamics of the phase transition process. The gel and liquid crystalline phases of DMPC coexisted over a broad temperature range (approximately 10 degrees C). Equal partitioning into two phases occurred at a transition temperature (Tm) of 28.5 degrees C. We developed a mathematical model to analyse AFM-derived DMPC membrane height changes as multi-peak Gaussian distributions. This approach allowed us to estimate the DMPC domain size, N, as 18-75 molecules per leaflet corresponding to a -4.2 nm diameter circular nanodomain. Lipid nanodomains may organize into microdomains or rafts which, in concert with proteins and other lipid components, play an important dynamic role in many biomedically important processes.
Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.
A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.
We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.
Dendrimer–platinate: a novel approach to cancer chemotherapy
- N Malik
- E G Evagorou
- R Duncan
Malik, N., Evagorou, E.G., Duncan, R., 1999. Dendrimer–platinate:
a novel approach to cancer chemotherapy. Anti-Cancer Drugs 10
(8), 767–776.
Dendrimer–platinate: a novel approach to cancer chemotherapy
- Malik