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Nucleic acid delivery with chitosan and its derivatives
Abstract
Chitosan is a naturally occurring cationic mucopolysaccharide. It is generally biocompatible, biodegradable, mucoadhesive, non-immunogenic and non-toxic. Although chitosan is able to condense nucleic acids (NA) (both DNA and RNA) and protect them from nuclease degradation, its poor water solubility and low transfection efficacy have impeded its use as an NA carrier. In order to overcome such limitations, a multitude of strategies for chitosan modification and formulation have been proposed. In this article, we will first give a brief overview of the physical and biological properties of chitosan. Then, with a special focus on plasmid DNA delivery, we will have a detailed discussion of the latest advances in chitosan-mediated NA transfer. For future research, the following three important areas will be discussed: chitosan-mediated therapeutic small RNA transfer, structure-activity relationships (SAR) in chitosan vector design, and chitosan-mediated oral/nasal NA therapy.
... The clinical application of gene and RNAi therapy requires a safe and efficient transport strategy [260]. The main delivery vehicles of nucleic acid therapy are presently based on viral and non-viral materials [261][262][263][264], and due to their more reliable security, non-viral delivery strategies have been widely used in nucleic acid delivery [262,265]. With the application of polyphenols in novel delivery systems, there are several studies available on the application of polyphenols in nucleic acid delivery systems to achieve effective delivery of polyphenols. ...
... The clinical application of gene and RNAi therapy requires a safe and efficient transport strategy [260]. The main delivery vehicles of nucleic acid therapy are presently based on viral and non-viral materials [261][262][263][264], and due to their more reliable security, non-viral delivery strategies have been widely used in nucleic acid delivery [262,265]. With the application of polyphenols in novel delivery systems, there are several studies available on the application of polyphenols in nucleic acid delivery systems to achieve effective delivery of polyphenols. ...
As naturally occurring bioactive products, several lines of evidence have shown the potential of polyphenols in the medical intervention of various diseases, including tumors, inflammatory diseases, and cardiovascular diseases. Notably, owing to the particular molecular structure, polyphenols can combine with proteins, metal ions, polymers, and nucleic acids providing better strategies for polyphenol-delivery strategies. This contributes to the inherent advantages of polyphenols as important functional components for other drug delivery strategies, e.g., protecting nanodrugs from oxidation as a protective layer, improving the physicochemical properties of carbohydrate polymer carriers, or being used to synthesize innovative functional delivery vehicles. Polyphenols have emerged as a multifaceted player in novel drug delivery systems, both as therapeutic agents delivered to intervene in disease progression and as essential components of drug carriers. Although an increasing number of studies have focused on polyphenol-based nanodrug delivery including epigallocatechin-3-gallate, curcumin, resveratrol, tannic acid, and polyphenol-related innovative preparations, these molecules are not without inherent shortcomings. The active biochemical characteristics of polyphenols constitute a prerequisite to their high-frequency use in drug delivery systems and likewise to provoke new challenges for the design and development of novel polyphenol drug delivery systems of improved efficacies. In this review, we focus on both the targeted delivery of polyphenols and the application of polyphenols as components of drug delivery carriers, and comprehensively elaborate on the application of polyphenols in new types of drug delivery systems. According to the different roles played by polyphenols in innovative drug delivery strategies, potential limitations and risks are discussed in detail including the influences on the physical and chemical properties of nanodrug delivery systems, and their influence on normal physiological functions inside the organism.
... Given the constraints in miRNA application due to their instability and delivery challenges, the progression of an effective delivery system like CS-NPs shows to be pivotal [26]. Chitosan has great potential in the delivery of polynucleotides because of its excellent biological qualities: it is biocompatible, biodegradable, mucoadhesive and non-toxic, bridging the gap between the therapeutic potential of miRNAs and their practical application [29,30]. ...
Background
Mutations in isocitrate dehydrogenase 1 and 2 ( IDH1 and IDH2 ), are present in most gliomas. IDH1 mutation is an important prognostic marker in glioma. However, its regulatory mechanism in glioma remains incompletely understood.
Results
miR-182-5p expression was increased within IDH1 -mutant glioma specimens according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples. (R)-2-hydroxyglutarate ((R)-2HG) treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p inhibition partially eliminated the oncogenic effects of R-2HG upon glioma cells. By direct binding to Cyclin Dependent Kinase Inhibitor 2 C ( CDKN2C ) 3’UTR, miR-182-5p inhibited CDKN2C expression. Regarding cellular functions, CDKN2C knockdown promoted R-2HG-treated glioma cell viability, suppressed apoptosis, and relieved cell cycle arrest. Furthermore, CDKN2C knockdown partially attenuated the effects of miR-182-5p inhibition on cell phenotypes. Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition in cell cycle check point and apoptosis markers. The engineered CS-NPs (antagomir-182-5p) effectively encapsulated and delivered antagomir-182-5p, enhancing anti-tumor efficacy in vivo, indicating the therapeutic potential of CS-NPs(antagomir-182-5p) in targeting the miR-182-5p/ CDKN2C axis against R-2HG-driven oncogenesis in mice models.
Conclusions
These insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/ CDKN2C axis, offering a promising therapeutic avenue against R-2HG’s oncogenic influence to glioma.
... For instance, lignosulfonate (LS), an anionic polymer with the components sulfonic acid and carboxyl and hydroxyl groups containing hydrophobic segments, was employed with chitosan to prepare chitosan/lignosulfonate composite micelles (CS-LS) through π-π and electrostatic interactions, displaying an average diameter of 239 nm [32]. With respect to the carrier design, chitosan-related nanocarriers facilitate delivery of specific agents, such as nucleic acid-loaded agents, due to the wealth of negative electric charges, and arduous efforts have been made by a myriad of scientists to seize the interaction [33,34]. Here, chitosan tends to be the ideal choice for burst co-delivery of siRNA and chemotherapeutics, alongside a further cationic modification with polyethyleneimine (PEI) to fetch a stronger electrostatic interaction than the naturally occurring polycationic chitosan [35]. ...
In the struggle against diseases, the development of nano-therapy has certainly been a tremendous progression owing to the various superiority, and chitosan is no doubt a kind of prominent biopolymer material with versatility for applications in disease treatments. For the rational construction of chitosan-related nano-biodevices, it is necessary to pay full attention to the material itself, where it is the material properties that guide the design criteria. Additionally, the well-matched preparation methods between material carriers and therapeutic agents draw much attention to the final construction since they seem to be more realistic. In detail, we present a comprehensive overview of recent advances in rational construction of chitosan-related nano-therapies with respect to material-property-oriented design criteria and preparation methods in the current review article, based on the foundation of continuous investigations. Based on this review, a portion of the various uses of chitosan-related nano-biodevices for biomedical applications are specifically discussed. Here, the strategies demonstrate the versatility of chitosan well, and the concept of being simple yet effective is well illustrated and vividly communicated. Altogether, a fresh concept concerning multi-functional chitosan and its derivative-related drug delivery systems for nano-therapy is proposed in this review, and this could be applied to other materials, which seems to be a novel angle.
... Polymeric vectors have potential as alternatives to lipids for RNA delivery owing to their wide chemical diversity, formulation flexibility, range of functional groups for targeting, stability in polyplex form, and well-established manufacturing processes 17 . Indeed, many different cationic polymers have been used as non-viral polymeric vectors for RNA delivery, including poly(ethyleneimine) 18 , poly(lysine) 19 , chitosan 20 , polyamidoamines 21 and poly(-aminoesters) 22 . ...
Therapeutic self-amplifying RNA (saRNA) is a promising approach for disease treatment, as it can be administered in lower doses than messenger RNA (mRNA) to achieve comparable protein production levels. However, saRNA requires an appropriate delivery vehicle to protect it during transit and facilitate its transfection. A widely-adopted approach has been to use polycations to condense these large anionic macromolecules into polyplex nanoparticles, however their high charge density often elicits cytotoxic effects. In this study we postulated that we could improve the potency and tolerability of such delivery vehicles by co-formulating poly(β-amino ester)s saRNA polyplexes with a non-toxic anionic polymer, γ-polyglutamic acid (γ-PGA) to neutralize partially this positive charge. Accordingly, we prepared a poly(β-amino ester) from 1,6-hexanedioldiacrylate (HDDA) and 4-aminobutanol (ABOL) and initially evaluated the physicochemical properties of the binary polyplexes (i.e. formed from polymer and saRNA only). Optimised binary polyplex formulations were then taken forward for preparation of ternary complexes containing pHDDA–ABOL, saRNA and γ-PGA. Our findings demonstrate that γ-PGA integration into polyplexes significantly enhanced transfection efficacy in HEK293T and A431 cells without affecting polyplex size. Notably, γ-PGA incorporation leads to a pronounced reduction in zeta potential, which reduced the toxicity of the ternary complexes in moDC, NIH3T3, and A431 cells. Furthermore, the presence of γ-PGA contributed to colloidal stability, reducing aggregation of the ternary complexes, as evidenced by insignificant changes in polydispersity index (PDI) after freeze–thaw cycles. Overall, these results suggest that incorporating the appropriate ratio of a polyanion such as γ-PGA with polycations in RNA delivery formulations is a promising way to improve the in vitro delivery of saRNA.
... Thus, the encapsulation and delivery of bacterial DNA also have great potential for research and application. As a positively charged natural polymer, chitosan can carry nucleic acids (RNA and DNA) and protect them from degradation by nucleases [76]. However, chitosan is rarely used as a carrier for nucleic acids due to its poor water solubility and low transfection efficiency. ...
A microencapsulated flame retardant PMDOPO was synthesized by solution blending method using EGDMA as initiator and AIBN as cross‐linking agent. Rigid polyurethane foams were prepared in a one‐step process. PMDOPO was incorporated into the foams by blending with polycarbonate diols (PCDL) and foaming additives. The results of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) showed that PMDOPO was well formed and uniformly dispersed in the foam. The pore size and density of the foams first increased and then decreased with the addition of PMDOPO. X‐ray photoelectron spectroscopy (XPS), vertical combustion test, and thermogravimetric analysis (TGA) showed that the PMDOPO content was directly proportional to the flame retardancy and the thermal stability. The major weight loss shifted to higher temperature intervals. The amount of residual char of RPUF/PMDOPO‐25 was enhanced 50 times to 10.63% compared to pure RPUF. In addition, SEM and Raman analyses of the combustion residual char revealed that a phosphorus‐containing char layer was formed during thermal degradation. The dense charcoal layer helps to insulate heat and suppress combustion. The universal testing machine showed that RPUF with 15 wt% content had the best compression resistance, with an enhancement of 1.8 MPa over pure RPUF.
... That enticed scientists to employ it in pharmaceuticals and biomedical research. However, it has poor water solubility and low transfection efficiency, making it unsuitable to be a carrier [132]. To surmount such hindrances, many strategies were proposed and developed until today for the modification and formulation of chitosan.Chitosan-based bionanocomposite is the most recognized element in this case that shows some outstanding performance to move a step ahead of the pharmaceutical and biomedical sectors than others [133]. ...
The complete dependency on synthetic and artificial materials threatens human life because they adversely affect our ecological system and affect the environment destructively. To mitigate that currently, the most talkative issue is to protect the environment by using biodegradable, nontoxic, biocompatible, and reusable materials for therapeutic, agricultural, food product manufacturing, and packaging purposes. In this case, chitosan-based materials show hopes, as chitosan is the second most plentiful natural biopolymer existing on the planet earth. Moreover, chitosan possesses film-forming abilities, antibacterial behavior, excellent mechanical and thermal properties. At present, chitosan-based bionanocomposite have appeared as ideal candidates for wide-ranging applications owing to the availability for surface modification together with their biodegradable, biocompatible, and nontoxic properties. However, this chapter highlights multiple aspects of chitosan such as its source, structural chemistry, properties, and modification. Additionally, It concentrates on chitosan-based bionanocomposites by describing its preparation techniques, characteristics, and applications. Finally, this chapter discusses the advantages and downsides of chitosan-based bionanocomposite to ensure its application superiority in numerous fields compared to other composites.
... Thus, the encapsulation and delivery of bacterial DNA also have great potential for research and application. As a positively charged natural polymer, chitosan can carry nucleic acids (RNA and DNA) and protect them from degradation by nucleases [76]. However, chitosan is rarely used as a carrier for nucleic acids due to its poor water solubility and low transfection efficiency. ...
Probiotics provide several benefits for humans, including restoring the balance of gut bacteria, boosting the immune system, and aiding in the management of certain conditions such as irritable bowel syndrome and lactose intolerance. However, the viability of probiotics may undergo a significant reduction during food storage and gastrointestinal transit, potentially hindering the realization of their health benefits. Microencapsulation techniques have been recognized as an effective way to improve the stability of probiotics during processing and storage and allow for their localization and slow release in intestine. Although, numerous techniques have been employed for the encapsulation of probiotics, the encapsulation techniques itself and carrier types are the main factors affecting the encapsulate effect. This work summarizes the applications of commonly used polysaccharides (alginate, starch, and chitosan), proteins (whey protein isolate, soy protein isolate, and zein) and its complex as the probiotics encapsulation materials; evaluates the evolutions in microencapsulation technologies and coating materials for probiotics, discusses their benefits and limitations, and provides directions for future research to improve targeted release of beneficial additives as well as microencapsulation techniques. This study provides a comprehensive reference for current knowledge pertaining to microencapsulation in probiotics processing and suggestions for best practices gleaned from the literature.
... The cationic nature of chitosan offers the advantage of carrying non-viral materials such as DNA for vaccination applications. Since nucleic acids have a strong negative charge, they can undergo electrostatic interaction with chitosan to form particulate entities known as polyplexes [173]. This interaction protects nucleic acids until they are delivered to the target site [169]. ...
Many recent studies focus on the pulmonary delivery of vaccines as it is needle-free, safe, and effective. Inhaled vaccines enhance systemic and mucosal immunization but still faces many limitations that can be resolved using polymeric nanoparticles (PNPs). This review focuses on the use of properties of PNPs, specifically chitosan and PLGA to be used in the delivery of vaccines by inhalation. It also aims to highlight that PNPs have adjuvant properties by themselves that induce cellular and humeral immunogenicity. Further, different factors influence the behavior of PNP in vivo such as size, morphology, and charge are discussed. Finally, some of the primary challenges facing PNPs are reviewed including formulation instability, reproducibility, device-related factors, patient-related factors, and industrial-level scale-up. Herein, the most important variables of PNPs that shall be defined in any PNPs to be used for pulmonary delivery are defined. Further, this study focuses on the most popular polymers used for this purpose.
... Chitosan as a natural cationic polysaccharide is also considered as a coating material for bacteria due to its biodegradability, low toxicity, and biocompatibility (Lai and Lin, 2009;Netsomboon and Bernkop-Schnurch, 2016). Cook et al. (2011) found that Bifidobacterium breve coated with chitosan and alginate showed more tolerant to the GI tract than that by single alginate coating, because that chitosan could stable alginate microcapsules and maintain the stability of probiotics in the stomach. ...
Therapeutic bacteria have shown great potential on anti-tumor therapy. Compared with traditional therapeutic strategy, living bacteria present unique advantages. Bacteria show high targeting and great colonization ability in tumor microenvironment with hypoxic and nutritious conditions. Bacterial-medicated antitumor therapy has been successfully applied on mouse models, but the low therapeutic effect and biosafe limit its application on clinical treatment. With the development of material science, coating living bacteria with suitable materials has received widespread attention to achieve synergetic therapy on tumor. In this review, we summarize various materials for coating living bacteria in cancer therapy and envision the opportunities and challenges of bacteria-medicated antitumor therapy.
... Chitosan derivatives are well reported for their use in delivery of poorly soluble drugs, for colon-targeted drug delivery, for mucosal drug delivery, ocular drug delivery and topical delivery [81][82][83][84]. ...
Chitosan has come a long way in biomedical applications: drug delivery is one of its core areas of imminent application. Chitosan derivatives are the new generation variants of chitosan. These modified chitosans have overcome limitations and progressed in the area of drug delivery. This review briefly surveys the current chitosan derivatives available for biomedical applications. The biomedical applications of chitosan derivatives are revisited and their key inputs for oral drug delivery have been discussed. The limited use of the vast chitosan resources for oral drug delivery applications, speculated to be probably due to the interdisciplinary nature of this research, is pointed out in the discussion. Chitosan-derivative synthesis and practical implementation for oral drug delivery require distinct expertise from chemists and pharmacists. The lack of enthusiasm could be related to the inadequacy in the smooth transfer of the synthesized derivatives to the actual implementers. With thiolated chitosan derivatives predominating the oral delivery of drugs, the need for representation from the vast array of ready-to-use chitosan derivatives is emphasized. There is plenty to explore in this direction.
... Thus, development of an efficient delivery system is crucial for successful delivery of these molecules. Chitosan can be considered a good candidate for functional delivery of polynucleotides, due to its high biocompatibility, biodegradability and low toxicity with a cationic potential that allows its interaction with the genetic materials and cell membrane [17,18]. On the other hand, chitosan is an intriguing candidate able to drive potent antigen-specific Th1 responses [19,20]. ...
Background and aim
MiR-155 could act as a key modulator of different aspects of immune system including Th1 responses. In this study, we designed chitosan nanoparticles containing miR-155-expressing plasmid and explored their effects as an adjuvant to enhance Th1 responses for potential future application against intracellular pathogens.
Methods
Nanoparticles were formulated by complex coacervation method and characterized for physicochemical and functional characteristics. Transfection efficiency in Raw 264.7 cells, effects on miR-155 target genes and NO production were evaluated. The prepared nanoparticles were co-administered as an adjuvant with ovalbumin to immunize mice and finally production of IFN-γ and IL-4 were measured by ELISA in splenocyte recall responses.
Results
The prepared nanoparticles had the mean size of 244 nm and zeta potential of +17 mV, respectively. Electrophoresis analysis indicated the high capability of nanoparticles to protect the plasmid from DNaseI degradation. Furthermore, nanoparticles showed an appropriate transfection efficiency in Raw 264.7 cells and could downregulate the expression of miR-155 target genes and also upregulate NO production. In vivo immunization examinations revealed successful shift of T cell responses toward Th1.
Conclusion
Our data suggests the high potential of chitosan nanoparticles containing miR-155-expressing plasmid as an adjuvant for significantly enhanced Th1-biased immune responses upon immunization with a given antigen.
... It is highly biocompatible, nontoxic, and biodegradable (86). It demonstrates a strong binding affinity with nucleic acids, cell surfaces, and mucous membranes due to the presence of several amines that impart a positive charge on it (87,88). Studies have shown that chitosan can function as an efficient adjuvant by inducing a type I IFN response by activating STING (89). ...
Respiratory viral pathogens like influenza and coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused outbreaks leading to millions of deaths. Vaccinations are, to date, the best and most economical way to control such outbreaks and have been highly successful for several pathogens. Currently used vaccines for respiratory viral pathogens are primarily live attenuated or inactivated and can risk reversion to virulence or confer inadequate immunity. The recent trend of using potent biomolecules like DNA, RNA, and protein antigenic components to synthesize vaccines for diseases has shown promising results. Still, it remains challenging to translate due to their high susceptibility to degradation during storage and after delivery. Advances in bioengineering technology for vaccine design have made it possible to control the physicochemical properties of the vaccines for rapid synthesis, heightened antigen presentation, safer formulations, and more robust immunogenicity. Bioengineering techniques and materials have been used to synthesize several potent vaccines, approved or in trials, against coronavirus disease 2019 (COVID-19) and are being explored for influenza, SARS, and Middle East respiratory syndrome (MERS) vaccines as well. Here, we review bioengineering strategies such as the use of polymeric particles, liposomes, and virus-like particles in vaccine development against influenza and coronaviruses and the feasibility of adopting these technologies for clinical use.
... 33,34 However, we and others have previously shown that forming complexes of siRNA with either of those polymers alone suffers from a lack of colloidal stability and show burst release of siRNA, thus limiting their applications. [35][36][37][38] A wide range of strategies have been suggested to overcome these limitations, including trimethylation, amino acid-conjugation, thiolation, glycosylation, pegylation, phospholipid-conjugation, aromatically modifications, and crosslinking methods, [39][40][41][42][43][44] with different rates of success. LBL assembled nano-drug delivery systems have been demonstrated as very promising platforms in drug delivery. ...
Excessive inflammatory responses in wounds are characterized by the presence of high levels of pro-inflammatory M1 macrophages rather than pro-healing M2 macrophages, which leads to delayed wound healing. Macrophage reprogramming from the M1 to M2 phenotype through knockdown of interferon regulatory factor 5 (irf5) has emerged as a possible therapeutic strategy. While downregulation of irf5 could be achieved by siRNA, it very much depends on successful intracellular delivery by suitable siRNA carriers. Here, we report on highly stable selenium-based layer-by-layer (LBL) nanocomplexes (NCs) for siRNA delivery with polyethyleneimine (PEI-LBL-NCs) as the final polymer layer. PEI-LBL-NCs showed good protection of siRNA with only 40% siRNA release in a buffer of pH = 8.5 after 72 h or in simulated wound fluid after 4 h. PEI-LBL-NCs also proved to be able to transfect RAW 264.7 cells with irf5-siRNA, resulting in successful reprogramming to the M2 phenotype as evidenced by a 3.4 and 2.6 times decrease in NOS-2 and TNF-α mRNA expression levels, respectively. Moreover, irf5-siRNA transfected cells exhibited a 2.5 times increase of the healing mediator Arg-1 and a 64% increase in expression of the M2 cell surface marker CD206+. Incubation of fibroblast cells with conditioned medium isolated from irf5-siRNA transfected RAW 264.7 cells resulted in accelerated wound healing in an in vitro scratch assay. These results show that irf5-siRNA loaded PEI-LBL-NCs are a promising therapeutic approach to tune macrophage polarization for improved wound healing.
... For example, chitosan, the most common cationic polysaccharide, has the ability to improve permeation of peptide drugs across mucosal epithelia (Thanou, Verhoef, & Junginger, 2001). It can encapsulate anionic nucleic acids or certain proteins and protect them from degradative enzymes (Lai & Lin, 2009). Moreover, chitosan bearing quaternized ammonium derivatives shows permanent positive charge, high solubility in a large range of pH values, and promising ability to enhance absorption of hydrophilic drugs (Thanou et al., 2001). ...
A new water-soluble ionocellulose material bearing imidazolium tosylate groups was synthesized in a two-step sequence from cellulose, and the electrical conduction properties of this bio sourced polyelectrolyte were investigated. First, tosylcellulose was prepared from neat cellulose and tosyl chloride in the ionic liquid 1-Butyl-3-methylimidazolium chloride [BMIM]Cl. Then, the ionocellulose containing imidazolium tosylate grafts was synthesized by reacting tosylcellulose and 1-methylimidazole. Both tosylcellulose and ionocellulose were characterized by different analytical techniques including elemental analysis, ¹³C NMR and FT-IR spectroscopy as well as thermogravimetric analysis. We then compared the dielectric relaxation of neat cellulose with ionocellulose. For both samples, two secondary relaxations were identified at low temperature. These two relaxations became faster in ionocellulose containing imidazolium tosylate grafts. At higher temperatures (T > room temperature), the conductivity is markedly dominated by ionic motions. This work opens new perspectives for preparing a new category of ionocellulose derivatives which are expected to find applications in energy storage technologies and for the elaboration as novel high-performance film dielectric capacitors.
... Therefore, the encapsulation and delivery of bacterial DNA has great potential. As a positively charged natural polymer, chitosan can entrap nucleic acids (NA) (both RNA and DNA) and protect them from degradation by nuclease (170). However, chitosan is rarely used as NA carrier because of its poor water solubility and low transfection efficiency. ...
The human intestine contains thousands of bacterial species essential for optimal health. Aside from their pathogenic effects, these bacteria have been associated with the efficacy of various treatments of diseases. Due to their impact on many human diseases, intestinal bacteria are receiving increasing research attention, and recent studies on intestinal bacteria and their effects on treatments has yielded valuable results. Particularly, intestinal bacteria can affect responses to numerous forms of immunotherapy, especially cancer therapy. With the development of precision medicine, understanding the factors that influence intestinal bacteria and how they can be regulated to enhance immunotherapy effects will improve the application prospects of intestinal bacteria therapy. Further, biomaterials employed for the convenient and efficient delivery of intestinal bacteria to the body have also become a research hotspot. In this review, we discuss the recent findings on the regulatory role of intestinal bacteria in immunotherapy, focusing on immune cells they regulate. We also summarize biomaterials used for their delivery.
... Zebra fish embryos are collected and rinsed several times using tap water. Healthy fertilized embryos are selected and pooled in the E3 medium [29]. The composition of the E3 medium is displayed in Table 1. ...
The polymer wrapped magnetic nanoparticles (Fe3O4) for the controlled release of drugs to the specific targeted site is one of the most effective way. In such a way, we have constructed a Fe3O4 magnetic nanoparticle wrapped chitosan (CS) polymer loaded with Ocimum tenuiflorum extract (phyto drug) for the drug delivery applications. The prepared composite nanomaterial is thoroughly characterized for understanding its crystal structure and surface morphology. Further, the zebra fish (Danio rerio) is chosen for the in vivo toxicity investigation of drug loaded Fe3O4/CS nanospheres. The phyto drug used has proven to have an excellent activity through our studies on zebra fish. Therefore, our designed drug loaded Fe3O4/CS nanospheres can be advocated for the real time diagnosis.
... Polymers are one group of materials that have been extensively used in the development of carriers for therapeutics delivery (Lai and He 2016;Lai and Shum 2015;Lai 2011Lai , 2013Lai and Lin 2009). In this chapter, we have introduced the use of polyanhydride, as well as lactide and glycolide polymers, in drug delivery. ...
In the previous chapter, the possible use of prodrug design in the enhancement of systemic delivery has been discussed. As the technique applicable to one drug may not be generalized to another drug, such an approach is comparatively labor intensive, and requires the structure of the agent designed to be manipulated case by case. As an alternative to, or as a complementary strategy for prodrug design, extensive efforts have been devoted to the development of diverse types of carriers over the last several decades. These carriers on one hand enable the delivery of multiple chemical entities and on the other hand allow for functionalization to enhance versatility and working performance. Due to their high structural flexibility, polymers have emerged as one of the most extensively studied materials for fabrication of such carriers. In this chapter, we will discuss different approaches to prepare polymeric particulates and will highlight the parameters to be characterized for optimal delivery performance in systemic delivery.
... 89 In addition, chitosan and its derivatives have the ability to bind nucleic acids, which presents the possible application of chitosan-mediated gene vector delivery in regenerative medicine. 90 For example, Guo et al. 91 utilized a chitosan/gelatin porous scaffold containing plasmid DNA encoding TGF-b1 to stimulate chondrocyte proliferation. ...
The meniscus plays a critical role in maintaining knee joint homeostasis. Injuries to the meniscus, especially considering the limited self-healing capacity of the avascular region, continue to be a challenge and are often treated by (partial) meniscectomy, which has been identified to cause osteoarthritis (OA). Currently, meniscus tissue engineering focuses on providing extracellular matrix-mimicking scaffolds to direct the inherent meniscal regeneration process, and it has been found that various stimuli are essential. Numerous bioactive factors present benefits in regulating cell fate, tissue development and healing but lack an optimal delivery system. More recently, bioengineers have developed various polymer-based drug delivery systems (PDDSs), which are beneficial in terms of the favorable properties of polymers as well as novel delivery strategies. Engineered PDDSs aim to provide not only an extracellular matrix-mimicking microenvironment but also the controlled release of bioactive factors with release profiles tailored according to the biological concerns and properties of the factors. In this review, both different polymers and bioactive factors involved in meniscal regeneration are discussed, as well as potential candidate systems, with examples of recent progress. This article aims to summarize drug delivery strategies in meniscal regeneration, with a focus on novel delivery strategies rather than on specific delivery carriers. The current challenges and future prospects for the structural and functional regeneration of the meniscus are also discussed.
... Over the years, different gel-based systems have been fabricated by using these polymers for biomedical use [5][6][7][8][9][10]. For example, the gel formed from a blend of chitosan and carboxymethyl cellulose sodium (CMC-Na) has been utilized for cutaneous administration of vitamin E to protect the skin from UV damage and to achieve moisturizing effects [11]. ...
Hydrogels are soft materials consisting of a three-dimensional network of polymer chains. Over the years, hydrogels with different compositions have been developed as drug carriers for diverse biomedical applications, ranging from cancer therapy and wound care to the treatment of neurodegenerative and inflammatory conditions. Most of these carriers, however, are designed only to deliver single agents. Carriers based on hydrogels for enabling co-delivery of multiple agents, with the release rate of each of the co-delivered agents to be tuned individually, are lacking. This study reports a one-pot method of fabricating alginate-based complex fibers with the Janus morphology, with carboxymethyl cellulose sodium as a polymeric modifier of the properties of each of the fiber compartments. By using malachite green and minocycline hydrochloride as model drugs, the generated fibers demonstrate the capacity of enabling the release profile of each of the co-delivered drugs to be precisely controlled. Along with their negligible toxicity and the retention of the activity of the loaded drugs, the complex fibers reported in this study warrant further development and optimization for applications that involve co-delivery of multiple agents.
... Unlike nanospheres, a therapeutic material in nanocapsules is confined in a cavity surrounded by a polymeric membrane [92][93][94]. Size and transfection efficiency of the nanoparticles are affected by various preparation methods including the concentration of therapeutic agents, pH, charge ratios and temperatures of preparation [95]. ...
Many drugs have been designed to treat diseases of the central nervous system (CNS), especially the neurodegenerative diseases. However, the presence of tight junctions at the blood-brain barrier has often compromised the efficiency of drug delivery to target sites in the brain. The principles of drug delivery systems across the blood-brain barrier are dependent on substrate-specific (i.e. protein transport and transcytosis) and non-specific (i.e. transcellular and paracellular) transport pathways, which are crucial factors in attempts to design efficient drug delivery strategies. This review describes how the blood-brain barrier presents the main challenge in delivering drugs to treat brain diseases and discusses the advantages and disadvantages of ongoing neurotherapeutic delivery strategies in overcoming this limitation. In addition, we discuss, the application of colloidal carrier systems, particularly nanoparticles, as potential tools for therapy for the CNS diseases.
... For instance, Mumper et al. firstly studied the potential of CS for in vitro pDNA delivery [83]. Through varying the N/P ratio (the ratio of CS nitrogens to DNA phosphates) and the M W of CS, nanopolyplexes (mean size ranging from 149 to 549 nm) with disparate spherical, annular, and toroidal topological conformations were observed [84]. ...
Low back pain stands as a pervasive global health concern, afflicting almost 80% of adults at some point in their lives with nearly 40% attributable to intervertebral disc degeneration (IVDD). As only symptomatic relief can be offered to patients there is a dire need for innovative treatments. Given the accumulating evidence that multiple microRNAs (miRs) are dysregulated during IVDD, they could have a huge potential against this debilitating condition. The way miRs can profoundly modulate signaling pathways and influence several cellular processes at once is particularly exciting to tackle this multifaceted disorder. However, miR delivery encounters extracellular and intracellular biological barriers. A promising technology to address this challenge is the vectorization of miRs within nanoparticles, providing both protection and enhancing their uptake within the scarce target cells of the degenerated IVD. This comprehensive review presents the diverse spectrum of miRs’ connection with IVDD and demonstrates their therapeutic potential when vectorized in nanomedicines.
https://authors.elsevier.com/c/1iexC_,OflnfMa
The remarkable impact of RNA nanomedicine during the COVID-19 pandemic has demonstrated the expansive therapeutic potential of this field in diverse disease contexts. In recent years, RNA nanomedicine targeting the liver has been paradigm-shifting in the management of metabolic diseases such as hyperoxaluria and amyloidosis. RNA nanomedicine has significant potential in the management of liver diseases, where optimal management would benefit from targeted delivery, doses titrated to liver metabolism, and personalized therapy based on the specific site of interest. In this review, we discuss in-depth the different types of RNA and nanocarriers used for liver targeting along with their specific applications in metabolic dysfunction-associated steatotic liver disease, liver fibrosis, and liver cancers. We further highlight the strategies for cell specific delivery and future perspectives in this field of research with the emergence of small activating RNA, circular RNA and RNA base editing approaches.
A novel strategy that has the potential to solve the drawbacks of the present conventional vaccines is the development of DNA vaccines. DNA vaccines offer a versatile and adaptable platform for treating a wide variety of diseases, as immunization targets may be easily adjusted by altering the gene sequences encoded in the plasmid DNA delivered. Due to their ability to elicit both humoral and cellular immune responses, their stability, and the ease with which they may be produced, plasmid DNA vaccines are quickly becoming the vaccine of choice, they are frequently safer than conventional vaccinations. Despite the highly encouraging outcomes of ongoing clinical trials, these vaccines' immunogenicity is compromised by a few factors. The use of various vaccine delivery techniques, the use of various polymer-based carriers, and the use of adjuvants are some of the several approaches that might be examined to better the immunogenicity of DNA vaccines made from plasmids. These advancements taken together might allow plasmid DNA vaccines to be successfully used in clinical settings.
mRNA therapy has shown great potential in infectious disease vaccines, cancer immunotherapy, protein replacement therapy, gene editing, and other fields due to its central role in all life processes. However, mRNA is challenging to pass through the cell membrane due to its significant negative charges and degradation from RNase, so the key to mRNA therapy is efficient packaging and delivery of it with appropriate vectors. Presently researchers have developed various vectors such as viruses and liposomes, but these conventional vectors are now difficult to meet the growing requirement like safety, efficiency, and targeting, so many novel delivery vectors with unique advantages have emerged recently. This review mainly introduces two categories of novel vectors: biomacromolecules and inorganic nanoparticles, as well as two novel methods of control and administration based on these novel vectors: controlled-release administration and non-invasive administration. These novel delivery strategies have the advantages of high safety, biocompatibility, versatility, intelligence, and targeting. This paper analyzes the challenges faced by the field of mRNA delivery in depth, and discusses how to use the characteristics of novel vectors and administrations to solve these problems.
Microfluidic mixing techniques are versatile and facile tools for improving reproducible production of pharmaceutics on one side and mimicking a complex cellular microenvironment on the other. In the first part of this chapter a summary of nanoparticulate drug delivery systems synthesized by microfluidic mixing is given. Different nanocarrier systems such as polymers, lipids, or a combinatorial approach, known as hybrid systems, are discussed in terms of advantages and disadvantages. The second part focuses on microfluidic systems as 3D cell culture models. The synergy of microfluidics and 3D cell culture enables mimicking of in vivo microenvironments while avoiding animal experiments. Complex organ-on-a-chip devices are accordingly developed to imitate human organs. The organ-on-a-chip platform facilitates novel drug screening possibilities. The ultimate goal of combining multiple organ-on-a-chip platforms would result in a human-body-on-chip, imitating the whole body, including several types of tissues and organs. This overall aim of microfluidic platforms is to improve patient outcomes by detecting individual diseases and discovering tailor-made therapies.
Gels with high drug release sustainability and intrinsic antibacterial properties are of high practical value for cutaneous drug administration, particularly for wound care and skin disease treatment. This study reports the generation and characterization of gels formed by 1,5-pentanedial-mediated crosslinking between chitosan and lysozyme for cutaneous drug delivery. Structures of the gels are characterized by using scanning electron microscopy, X-ray diffractometry and Fourier-transform infrared spectroscopy. An increase in the mass percentage of lysozyme leads to an increase in the swelling ratio and erosion susceptibility of the resulting gels. The drug delivery performance of the gels can be changed simply by manipulating the chitosan/lysozyme mass-to-mass ratio, with an increase in the mass percentage of lysozyme leading to a decline in the encapsulation efficiency and drug release sustainability of the gels. Not only do all gels tested in this study show negligible toxicity in NIH/3T3 fibroblasts, they also demonstrate intrinsic antibacterial effects against both Gram-negative and Gram-positive bacteria, with the magnitude of the effect being positively related to the mass percentage of lysozyme. All these warrant the gels to be further developed as intrinsically antibacterial carriers for cutaneous drug administration.
Purpose:
Magnetic nanoparticles have been used in diverse pharmaceutical applications because they can potentially be used to target specific sites. In the present work, a new type of nanocomposites is designed as a carrier of controlled bioactive agent delivery.
Methods:
Amine-functionalized magnetic nanoparticles (amine-MNPs) are coupled with carboxymethyl chitosan (CMC) to generate the nanocomposites, namely MNPs-CMC, which can be further loaded with doxorubicin (DOX) to produce MNPs-CMC-DOX. The generated nanocomposites are characterized by using various techniques (including FTIR, 1H-NMR, DSC, TGA, SEM, TEM and XRD). In vitro drug release studies are conducted in PBS with different pH values (1.2 and 6.8) at different temperatures (25°C and 37°C). The toxicity of the nanocomposites is tested in MCF-7 and 3T3 cells. The ROS-generating capacity of the nanocomposites is determined in treated cells using 2',7'-dichlorodihydrofluorescein diacetate.
Results:
The structures of MNPs, CMC, and nanocomposites are confirmed by FTIR, XRD, and 1H-NMR data reveals the formation of CMC from chitosan (CS). The size of MNPs is estimated by TEM to be around 25 nm. After conjugation with CMC, the size of the nanocomposites increases to 46-57 nm. Based on the release profiles of MNPs-CMC-DOX, our nanocomposites are pH-responsive. In addition, our nanocomposites show reactive oxygen species (ROS)-generating capacity and cell type-dependent toxicity.
Conclusion:
Our nanocomposites show high potential for use in bioactive agent delivery. Along with their ROS-generating capacity, they warrant further development as pH-responsive carriers for therapeutic applications.
Clusteroluminescence is a phenomenon caused by the aggregation of non-conjugated electron-rich units, leading to an increase in the emission efficiency when the material is in aggregated or solid states. Because many of the non-aromatic polymers with clusterization-triggered emission features have good biocompatibility and biodegradability, along with their good processability for formulation and sustained release of a diverse range of bioactive agents, they have emerged as a new class of materials for the development of optically active carriers over the last several decades. Despite this, serious discussions on the emerging yet encouraging potential of clusteroluminogenic polymers in bioactive agent delivery have been scant in the literature till now. The objective of this article is to fill this gap by reviewing the current status of the development and use of clusteroluminogenic polymers in carrier design. It is hoped that this article can not only offer insights into the effective use of non-aromatic clusteroluminogenic polymers in carrier development but can also serve as a trigger for innovation to enhance the functionality and versatility in bioactive agent delivery research.
Chitosan is a natural biopolymer with a high potential to serve as a carrier for drug and gene delivery. Several types of chitosan with and without modifications were reported for the delivery of gene therapeutic agents. Chitosan‐based materials are biocompatible, condense effectively with oligonucleotides, are cheap, and can be transfected in several types of cells. The chemistry of chitosan, such as functional groups, enables structural modification to satisfy the requirements prior to use for the synthesis of an effective delivery system. This chapter introduces the applications of chitosan as a carrier for gene delivery. It covers the basic chemistry of chitosan and their modifications. Applications of chitosan as a carrier for gene delivery and the advantages and disadvantages of the chitosan‐based carrier are summarized. This chapter is useful for researchers and scientists interested in the applications of chitosan as a carrier for gene‐based therapeutic agents.
Breast cancer is one of the most frequently diagnosed cancers that is threatening women’s life. Current clinical treatment regimens for breast cancer often involve neoadjuvant and adjuvant systemic therapies, which somewhat are associated with unfavorable features. Also, the heterogeneous nature of breast cancers requires precision medicine that cannot be fulfilled by a single type of systemically administered drug. Taking advantage of the nanocarriers, nanomedicines emerge as promising therapeutic agents for breast cancer that could resolve the defects of drugs and achieve precise drug delivery to almost all sites of primary and metastatic breast tumors (e.g. tumor vasculature, tumor stroma components, breast cancer cells, and some immune cells). Seven nanomedicines as represented by Doxil® have been approved for breast cancer clinical treatment so far. More nanomedicines including both non-targeting and active targeting nanomedicines are being evaluated in the clinical trials. However, we have to realize that the translation of nanomedicines, particularly the active targeting nanomedicines is not as successful as people have expected. This review provides a comprehensive landscape of the nanomedicines for breast cancer treatment, from laboratory investigations to clinical applications. We also highlight the key advances in the understanding of the biological fate and the targeting strategies of breast cancer nanomedicine and the implications to clinical translation.
Food packaging can extend the shelf life of food products and enhance the safety and quality of the food. This study reports food-grade polyelectrolyte complex films generated via electrostatic interactions between two cellulose-based agents [viz., hypromellose-graft-chitosan, and carmellose sodium]. At optimal conditions, our films show good barrier properties, high transparency, and high efficiency in post-production agent loading. They also demonstrate intrinsic antibacterial effects against both Gram-negative and Gram-positive bacteria. By using frozen chicken breasts as a model, the films enable real-time monitoring of the status of the frozen food due to the property of clusterisation-triggered emission. Along with their negligible toxicity, our films warrant further development as multi-functional films for effective and self-indicating food packaging.
The present study reports the generation of 2-hydroxyethyl starch microparticles for co-delivery and controlled release of multiple agents. The obtained microparticles are characterized by using Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction analysis, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. By using ofloxacin and ketoprofen as drug models, the release sustainability of the microparticles is examined at pH 1.2, 5.4, and 6.8 at 37 oC, with Fickian diffusion being found to be the major mechanism controlling the kinetics of drug release. Upon being loaded with the drug models, the microparticles show high efficiency in acting against Escherichia coli and Bacillus cereus. The results suggest that our reported microparticles warrant further development for applications in which co-administration of multiple bioactive agents is required.
The unique physico-chemical properties of cationic polymers and their ability to be easily modified make them attractive for many biological applications. As a result there is a vast amount of research focussed on designing novel natural or synthetic cationic polymers with specific biological functionality.
Cationic Polymers in Regenerative Medicine brings together the expertise of leading experts in the field to provide a comprehensive overview of the recent advances in cationic polymer synthesis, modification and the design of biomaterials with different structures for therapeutic applications. Chapters cover recent developments in novel cationic polymer based systems including poly(L-lysine), Poly(N,N-dimethylaminoethyl methacrylate) and cationic triazine dendrimers as well as cationic polymer-coated micro- and nanoparticles and cationic cellulose and chitin nanocrystals. Applications discussed in the book include drug and gene delivery, therapeutics in thrombosis and inflammation as well as gene therapy.
Suitable both for an educational perspective for those new to the field and those already active in the field, the book will appeal to postgraduates and researchers. The broad aspects of the topics covered are suitable for polymer chemists interested in the fundamentals of the materials systems as well as pharmaceutical chemists, bioengineering and medical professionals interested in their applications.
Mesoporous silica nanoparticles coated with Chitosan are exploited here as a potential carrier for oral vaccine delivery. Bovine serum albumin (BSA) was used as a protein antigen model to reveal the carrier property. Chitosan‐coated BSA‐loaded silica NPs had particle size 345 ± 60 nm with a cationic surface charge of 18.28 ± 0.71 mV. The encapsulation efficiency, drug loading was 25.34 ± 0.76 and 20.21 ± 0.48%, respectively. Transmission electron microscopy investigation showed the spherical shape of NPs, also confirmed surface coating around modified nanoparticles (NPs), and nitrogen absorption/desorption isotherm confirmed mesostructured inside the NPs. Fourier transform infrared spectroscopy did not show any physiochemical interactions between excipients and formulations. The structural stability of antigen after release from NPs was confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis, and chitosan‐coated silica NPs exhibited a slow‐release pattern. The results of in vivo experiments presented that chitosan‐mesoporous silica NPs could induce a robust immune response in mice, indicating that chitosan‐mesoporous silica NPs might be used as a promising carrier for oral vaccine delivery.
Diverse nanoparticulate systems, ranging from polymeric nanoparticles to liposomes, have been exploited as carriers of bioactive agents in recent years; however, the use of these systems has been confined largely to the laboratory context till now. Systems that can successfully be adopted for bioactive agent delivery in practice are few. Herein, such low efficiency in clinical translation is partly due to the lack of awareness of the similar nature between a carrier and a real drug. To rectify this situation, it is important to treat a carrier as an ordinary drug despite its absence of therapeutic effects. The current situation in prevalent bioactive agent delivery research, as well as those routine research practices that should be changed to enhance clinical translation, will be discussed here. Diverse nanoparticulate systems have been exploited as carriers of bioactive agents over the years; however, systems that can finally get to real applications are few. Such low efficiency in clinical translation is partly due to the lack of awareness of the similar nature between a carrier and a real drug.
Nanomaterials are excellent drug delivery systems, yet, they must be functionalized in a manner compatible with the biological environment. Regarding delivery of the payloads, it is critical to monitor the nanocarrier’s biocompatibility and the ability to control its drug encapsulation and release, as well as targeting. The current challenges include avoiding negative host immune responses, optimizing stability in biological environments, and achieving precise interactions with the targets. Contemporary advances in structural DNA nanotechnology, DNA origami, and supramolecular DNA assembly make it possible to produce complex multi-functional DNA nanostructures, wherein precise control of the size, geometry, and appearance of the ligands is feasible. DNA nanostructures offer ease of synthesis and conjugation of functional moieties to target the release of cargo or to analyze important biomarkers for diagnostics. Furthermore, the biocompatibility, programmability, responsiveness to biomolecules, cell-surfaces, and organisms make such DNA nanomaterials highly suitable for potential translational applications. This overview summarizes the recent developments in functionalizing, stabilizing and applying DNA nanostructures for potential biomedical applications.
Keywords:
Biological material; drug delivery; DNA nanostructures; diagnostics; biomedicine.
Graphene possesses a large specific surface area, a high Young's modulus, high fracture strength, high electrical conductivity, and excellent optical performance. It has been widely studied for biomedical use since its first appearance in the literature. This article offers an overview of the latest advances in the design of graphene-based materials for delivery of bioactive agents. To enhance the translation of these carriers into practical use, the toxicity involved is needed to be examined in future research in more detail. In addition, guidelines for standardizing experimental conditions during the evaluation of the performance of graphene-based materials are required to be established so that candidates showing higher practical potential can be more effectively identified for further development. This can streamline the optimization and use of graphene-based materials in delivery applications.
A series of linear polyester-based, α-cyclodextrin (CD)-threaded polyrotaxanes (PRTx) were synthesized for siRNA delivery. The investigation into the effect of the presence of polyester linkages in polyrotaxane established the structural-activity relationship between polyrotaxane and siRNA transfection efficiency. The ester-based polyrotaxane exhibited higher threading efficiency than poly(ethylene glycol)-based polyrotaxane. The threading efficiency is the driving force for transfection, as it regulates the positive charge density on polyrotaxane. Polyester-based polyrotaxane formed stable and effective transfection nanoplexes with siRNA at lower N/P ratios, signifying the high gene loading capacity of the developed supramolecular vectors. Our findings suggest that biochemical properties of the transfection complexes depend on the structure of the axis and threading efficiency of polyrotaxane, which further influences the transfection efficiency. The enhanced gene silencing efficiency and safety are compared with those of extensively explored poly(ethylene glycol)-based polyrotaxane, polyethyleneimine (a gold standard), and lipofectamine (a commercial standard), which are used as siRNA delivery vectors.
Sonodynamic therapy (SDT) by utilizing ultrasonic waves triggers the generation of reactive oxygen species (ROS) with the help of sonosensitizers to destruct deep‐seated tumors has attracted great attention. However, the efficacy of SDT may not be robust enough due to the insufficient oxygen supply within solid tumors. Additionally, repeated injections and treatments, which are often required to achieve the optimal therapeutic responses, may cause additional side effects and patient incompliance. Herein, a thermo‐triggered in situ hydrogel system is developed in which catalase (CAT) conjugated with sonosensitizer meso‐tetra (4‐carboxyphenyl) porphine (TCPP) is mixed into chitosan (CS) and beta‐glycerol phosphate disodium (GP) to form the precursor solution. After injection of the precursor solution into tumors, the in situ sol–gel transformation will occur as triggered by the body temperature, resulting in the localized tumor retention of TCPP‐CAT. The locally restrained TCPP‐CAT not only produces ROS under ultrasonic treatment, but also sustainably reverses the oxygen‐deficient status in solid tumors by triggering the O2 generation from the decomposition of endogenous H2O2, further promoting the efficacy of SDT. As a result, the repeated SDT after a single dose injection of such a hydrogel can offer robust treatment effects to effectively eradicate tumors.
Biofluorescence in living entities is a functional process associated with information conveyance; whereas the capacity to respond to internal physiological signals is a unique property of a cell. By integrating these two biological features into materials design, a bioinspired material, namely CPS, is developed. Contrary to conventional luminescent polymeric systems whose emission comes from π‐conjugated structures, this material displays clusterization‐triggered emission. In the preclinical trial on a dermal punch model of tissue repair, it successfully increases the rate of wound closure, reduces inflammatory cell infiltration, and enhances collagen deposition. It can also relay changes in internal chemical signals into changes in its intrinsic luminescence for biphasic chemical sensing to prevent possible occurrence of skin hyperpigmentation caused by minocycline hydrochloride in wound therapy. Together with its ease of fabrication, high biocompatibility, high drug loading efficiency, and high release sustainability, CPS shows high potential to be developed into an intelligent solid‐state device for wound treatment in the future.
Bacterial therapy, which presents a smart platform for delivering and producing therapeutic agents, as monotherapy or in combination with other therapeutic modes, has provided a breakthrough for the treatment of a range of diseases. The integration of synthetic biology technology with bacteria enables their characteristics like chemotaxis and biomolecule secretion to outperform conventional diagnostics and therapeutics, thereby facilitating their clinical applications in a range of diseases. Compared to injection-administered bacteria, orally-delivered bacteria improve patient compliance while avoiding the risk of systemic infections. However, oral administration of microbes always leads to a substantial loss of viability due to the highly acidic environment in the stomach and bile salt in the intestine. Thus, the formulation of these bacteria into microcapsules using appropriate biomaterials is a promising approach for reducing cell death during gastrointestinal passage and controlling the release of these therapeutic cells across the intestinal tract. In this review, we reveal the basic principles of oral bacterial delivery, from internal genetic engineering approaches to external encapsulation and modification, and summarize the most recent biomedical applications. Finally, we discuss future trends in oral bacterial therapy as well as current challenges that need to be resolved to advance their clinical applications.
Luminescence plays an important role in advancing various research fronts. Traditionally, luminescence from polymeric luminogens is attributed to the presence of conjugated structures. In recent years, efforts have, however, been directed to deciphering the photophysics of polymeric luminogens in which conjugated structures are absent. These non-conjugated polymers do not experience aggregation-caused quenching as the conventional ones do. Instead, they display aggregation induced emission (AIE)-like properties, thereby exhibiting potential for use in drug delivery because carriers in many dosage forms (including tablets, microgels, nanoparticles, and films) have to exist in a highly aggregated state or solid state. The objectives of this article are to highlight the practical potential of non-conjugated polymeric luminogens as intrinsically luminescent carriers for drug delivery, and to revisit the opportunities and challenges of these carriers for future research in theranostics.
Conventional methods for gene delivery with non-viral or viral delivery carriers are beset with various disadvantages such as immune reactions, low transfection efficiency, and toxicity. Innumerable methods are under investigation to help improve the development of biodegradable polymers with low cytotoxicity, transcellular transport ability, favorable physicochemical properties, ease of modification by targeting ligands, and high transfection efficiency. Chitosan is a biodegradable polymer that has attained a lot of attention as a gene delivery vector due to its ease of modification, high transfection efficiency, and exceptional biocompatibility. Chitosan being cationic in nature can form polyelectrolyte complexes with negatively charged DNA allowing nucleic acid condensation along with protection from nucleases, which is widely beneficial in gene therapies. Moreover, factors such as pH, degree of acetylation, N/P ratio, and surface modifications can be suitably investigated to improve transfection efficiency of chitosan-based vectors. Various chitosan-based gene delivery systems developed in the past decade including chitosan-based polyplexes, nanoparticles, and DNA vaccines have been discussed in this chapter. The goal of this book chapter is to review recent advancements in gene therapy with major focus on chitosan and its applications as a gene delivery vector.
Theranostics refers to the incorporation of therapeutic and diagnostic functions into one material system. An important class of nanomaterials exploited for theranostics is metal nanoclusters (NCs). In contrast to gold and silver NCs, copper is an essential trace element for humans. It can be more easily removed from the body. This, along with the low cost of copper that offers potential large‐scale nanotechnology applications, means that copper NCs have attracted great interest in recent years. The latest advances in the design, synthesis, surface engineering, and applications of copper NCs in disease diagnosis, monitoring, and treatment are reviewed. Strategies to control and enhance the emission of copper NCs are considered. With this synopsis of the up‐to‐date development of copper NCs as theranostic agents, it is hoped that insights and directions for translating current advances from the laboratory to the clinic can be further advanced and accelerated. Copper nanoclusters are an emerging class of fluorophores which can be synthesized using “top‐down” or “bottom‐up” approaches. Their emissive and physiological performance can be enhanced by using various techniques, ranging from surface engineering to size manipulation, for theranostic applications.
This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio ( Rg / RH ) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.
Nasal drug administration has been used as an alternative route for the systemic availability of drugs restricted to intravenous administration. This is due to the large surface area, porous endothelial membrane, high total blood flow, the avoidance of first-pass metabolism, and ready accessibility. The nasal administration of drugs, including numerous compound, peptide and protein drugs, for systemic medication has been widely investigated in recent years. Drugs are cleared rapidly from the nasal cavity after intranasal administration, resulting in rapid systemic drug absorption. Several approaches are here discussed for increasing the residence time of drug formulations in the nasal cavity, resulting in improved nasal drug absorption. The article highlights the importance and advantages of the drug delivery systems applied via the nasal route, which have bioadhesive properties. Bioadhesive, or more appropriately, mucoadhesive systems have been prepared for both oral and peroral administration in the past. The nasal mucosa presents an ideal site for bioadhesive drug delivery systems. In this review we discuss the effects of microspheres and other bioadhesive drug delivery systems on nasal drug absorption. Drug delivery systems, such as microspheres, liposomes and gels have been demonstrated to have good bioadhesive characteristics and that swell easily when in contact with the nasal mucosa. These drug delivery systems have the ability to control the rate of drug clearance from the nasal cavity as well as protect the drug from enzymatic degradation in nasal secretions. The mechanisms and effectiveness of these drug delivery systems are described in order to guide the development of specific and effective therapies for the future development of peptide preparations and other drugs that otherwise should be administered parenterally. As a consequence, bioavailability and residence time of the drugs that are administered via the nasal route can be increased by bioadhesive drug delivery systems. Although the majority of this work involving the use of microspheres, liposomes and gels is limited to the delivery of macromolecules (e.g., insulin and growth hormone), the general principles involved could be applied to other drug candidates. It must be emphasized that many drugs can be absorbed well if the contact time between formulation and the nasal mucosa is optimized.
Chitosan, a polysaccharide with structural characteristics similar to glycosamino glycan, was studied for various biomedical applications including membranes in hemodialysis, artificial skin or bioactive wound dressings, as an excipient in drug delivery systems, as a matrix in perfusion columns and as a material possessing hemostatic potential to promote nerve growth. Chitosan is, in fact, prescribed for obesity since it is an excellent fat binder. With these multiple applications, chitosan - a biodegradable and naturally available material - will emerge as a household name and as an important biomedical material of the 21st century. In this paper, we aim to present the various drug delivery applications of this interesting biopolymer.
We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 103 NLS-bound pCMVL copies. With 104 copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p
We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-l-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-l-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-l-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-l-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-l-lysine conjugate were prevented by neurotensin (1 μM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-l-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-l-lysine conjugate could be a useful tool for gene delivery to those neuronal systems.
Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.
We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant
c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328
(PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that
distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP;
peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained
nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin
and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear
accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear
localization signal of c-myc, and M2 serves some other and essential function.
The adenovirus E1a gene products are nuclear proteins important in transcriptional control of viral functions during infection.
By producing normal E1a proteins and derivatives of E1a in bacteria and microinjecting these proteins into cultured cells,
we were able to examine their ability to localize to the nucleus. We showed that a short peptide sequence at the carboxyl
terminus of E1a is necessary for the rapid (30-min) nuclear localization of that protein. Additionally, we showed that just
the last five amino acids of E1a are sufficient to direct nuclear accumulation of a heterologous protein, Escherichia coli
galactokinase, with the same kinetics as native E1a. The mechanism by which this pentamer mediates rapid nuclear localization
was examined by testing the ability of a galactokinase derivative which has no signal pentamer to exit the nucleus, as well
as to enter it. Because neither free entry nor exit was detected, the effect of the signal is unlikely to be through increased
nuclear retention of freely diffusible proteins but rather by enhancement of entry into the nucleus.
Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.
Various acetylated chitosan derivatives and mixtures of chitin and chitosan, covering the range of the degree of deacetylation (DDA) from 0-100% were analyzed by 1H-NMR spectroscopy and infrared spectroscopy. The use of the 1070 cm-1 or 1030 cm-1 absorption band as an internal standard in the determination of DDA from the absorbance of the amide I bands at 1655 cm-1 and 1630 cm-1 or the amide II band at 1560 cm-1 was studied. There is a good correlation between the results from IR spectroscopy and those from 1H-NMR spectroscopy.
Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.
Ten pyrimidine nucleoside analogues, including (B)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5' phosphotransferase, EC 2.7.2.21) gene. (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyluracil (IVFAU) and (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. Inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-beta-D-arabinofuranosylpurine (araM), and at least five- to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.
Non-viral gene delivery systems have the potential to create viable pharmaceuticals from nucleic acids. These DNA delivery systems contain lipids and/or cationic polymers. In order for these systems to be developed into commercial products, several barriers must be overcome. These include obstacles in manufacturing, formulation and stability. In vivo, problems of extracellular non-specific interactions and intracellular trafficking to the nucleus are also encountered. Recent progress has been made in overcoming these issues.
It has recently become very important to develop drug delivery systems for water-soluble natural macromolecules, such as peptides, proteins and polynucleotides. We have found that poly(ethylene glycol)grafted chitosan, PEG-g-chitosan, formed nanoparticles through intermolecular hydrogen bonding in an aqueous solution. PEG-g-chitosan nanoparticles can be expected to incorporate water-soluble, polar or anionic molecules, which can then interact with chitosan by hydrogen bonds or electrostatiscally. We therefore decided to investigate the incorporation of a peptide hormone, insulin, as a model peptide drug into PEG-g-chitosan nanoparticles. PEG-g-chitosan nanoparticles incorporated a certain quantity of insulin molecules spontaneously, but this depended on the degree of introduction of PEG chain on chitosan. The release rate also depended on the degree of introduction of PEG chain on chitosan. The sustained release of insulin from nanoparticles was also observed. PEG-g-chitosan nanoparticles are can be expected to be applied as delivery vehicles for peptide drugs.
Nonviral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors. Chitosan is considered to be a good candidate for the gene delivery system since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic charge potential. However, the use of chitosan for gene delivery is limited due to low transfection efficiency. To enhance the transfection efficiency, water-soluble chitosan (WSC) was coupled with urocanic acid (UA) bearing imidazole ring which can play the crucial role in endosomal rupture through proton sponge mechanism. The urocanic acid-modified chitosan (UAC) was complexed with DNA, and UAC/DNA complexes were characterized. The sizes of UAC/DNA complexes under physiological condition (109–342 nm) were almost same as those of chitosan–DNA complexes. UAC also showed good DNA binding ability, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coupling with UA and increased with an increase of UA contents in the UAC.
Purpose. Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1–3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium.
Methods. Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule 14C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity.
Results. Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of 14C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship.
Conclusions. The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.
The degree of acetylation of chitosan can be determined in acetic acid solutions (∼0·01m) containing 1 g dry chitosan per litre by first derivative ultraviolet spectrophotometry at 199 nm. At this wavelength, the N-acetylglucosamine absorbance readings are linearly dependent on concentration and are not influenced by the presence of acetic acid. Correction factors for the contribution of glucosamine in highly deacetylated chitosans can be easily derived. Typical results for the chitosan of Euphausia superba are: degree of acetylation, 42·6; relative standard deviation, 1·3%; confidence limits, ±0·7. This method is simpler, more precise and faster than the infrared method. Sonication of chitosan solutions leads to immediate chain degradation and to detectable deacetylation after more prolonged periods of time, especially when the pH is 1·0.
Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting `the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP>4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (HTS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements.
An N-dodecylated chitosan (CS-12) was synthesized from dodecyl bromide and chitosan and was assembled with DNA to form a polyelectrolyte complex (DNA/CS-12 PEC). UV was used to examine the thermal stability of DNA embedded in PEC. The results indicate that the incorporation of dodecylated chitosan can enhance the thermal stability of DNA. The analysis of AFM image shows that PEC develops a globule-like structure composed of 40–115 DNA molecules. Dissociation of PEC was investigated by the addition of low molecular weight electrolytes. The added small molecular salts dissociate the PEC, inducing DNA to release. The ability of Mg2+ to dissociate PEC is greater compared to that of Na+ and K+. From AFM images, it can be visualized that DNA remains intact and undamaged due to the protection from DNase offered by alkylated chitosan. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 3391–3395, 2001
The composition and sequence of 2-acetamino-2-deoxy-β-d-glucose (GlcNAc) and 2-amino-2-deoxy-β-d-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogenous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degre eof acetylation of 50% were soluble at neutral pH.
In studying drug absorption from the nasal mucous membrane, it is essential to have a clear understanding of anatomy and physiology of the nose, and how it relates to the characteristics of the delivery system used. The human nose is characterized by an individually varying shape and caliber, which might interfere with standard recommendations of intranasal medication. It is also of significance that there is a tendency for reflex- induced and profuse watery hypersecretion from glands, and for quick and considerable changes of mucosal thickness due to the presence of large venous sinusoids. These are factors which can interfere with pharmacokinetics. Also mucociliary transport rate has to be taken into consideration, as the drug is removed from the absorptive mucous membrane within 30 min. Intranasal drug distribution has been poorly studied in relation to nasal anatomy and pathology. In contrast to common believe, nasal inflammation does not seem to increase drug absorption. On the contrary, blockage, sneezing and rhinorrhoea might preclude the absorption.
Three commercial chitosans varying in average degree of acetylation (DA) were characterized in terms of average molar massesMn,Mw, the second virial coefficientB and radius of gyrationRgz, and intrinsic viscosities by membrane osmometry, static light scattering, and capillary viscometry in acetate buffer of pH 4.5. To obtain satisfactory light scattering data, the method used for clarification was of great importance. Combined ultracentrifugation and membrane filtration was found to be the method of choice. Using the model for wormlike chains with excluded volume and a logarithmic molar mass distribution, the persistence lengthLp was determined asLp = 6 nm. No concentration-dependent association was observed for the low concentrations studied. Samples were also fractionated on Sepharose CL-2B for subsequent light scattering and viscosity measurements to establish relationships betweenMwand [η] andRg, respectively. These relationships were found to be independent of the DA for the bulk of molecularly dispersed chitosan. Model calculations aimed at checking the consistency of data led, as to be expected, to an increasing excluded volume effect with increasing molar mass and steadily increasing polydispersities of the fractions with increasing elution volume. The levelling off of the [η]-Mw andRg-Mg relations for fractions of increasingly high molar mass was assigned to chemical heterogeneity of samples in terms of DA distributions.
Chitosan microspheres were prepared by a novel precipitation process using sodium sulfate as precipitant. Low, medium, and high molecular weight chitosan was chosen for the formulation of microspheres. The extent of precipitation was controlled by the concentration of sodium sulfate and monitored by turbidity measurement. The amount of sodium sulfate required for the preparation of the microspheres depended on the molecular weight of chitosan. The particle size was determined by photon correlation spectroscopy (PCS) and centrifugal sedimentation. The morphological characteristics were examined using a scanning electron microscope (SEM). The surface charge was measured by microelectrophoresis. After preparation the loading property with various anti-inflammatory drugs was investigated using spectrophotometry. The influence of surface adsorption on the drug modification was controlled by differential scanning calorimetry (DSC). Drug liberation was tested in vitro using side-by-side diffusion cells with a dialysis membrane made of cellulose acetate. The highest loading (up to 30.5% relative to the polymer mass) was achieved with prednisolone sodium phosphate (PSP). The adsorbed drug was present in an amorphous form. The drug release from the microspheres was dependent on the drug-polymer ratio.
Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 μg/ml, the size of particles was optimized to ∼100–250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by Lipofectamine™-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.
Due to recent advances, numerous bioactive peptides are now available in large quantities. Administering these substances by the oral route appears as a formidable challenge due to their insufficient stability in the gastrointestinal tract and their poor absorption pattern. Several approaches have been investigated to improve their oral bioavailability. Among them, the use of polymeric particulate delivery systems (microparticles and nanoparticles) represents a promising concept. Encapsulating or incorporating peptides in particles should at least protect these substances against degradation and, in some cases, also enhance their absorption. The aim of this paper is to review the principal studies where peptide-loaded particles were administered by the oral route. The preparation methods and in vitro trials are presented and in vivo results are discussed with emphasis placed on the peptide blood levels reached or on the biological effects observed. Whether or not intact particles can be taken up and translocated to the systemic circulation is not the aim of this review.
We report a novel approach to grow highly oriented, freestanding and structured carbon nanotubes (CNTs) between two substrates, using microwave plasma chemical vapour deposition. Sandwiched, multi-layered catalyst structures are employed to generate such structures. The as-grown CNTs adhere well to both the substrate and the top contact, and provide a low-resistance electric contact between the two. High-resolution scanning electron microscope (SEM) images show that the CNTs grow perpendicular to these surfaces. This presents a simple way to grow CNTs in different, predetermined directions in a single growth step. The overall resistance of a CNT bundle and two CNT-terminal contacts is measured to be about 14.7 k Ω. The corresponding conductance is close to the quantum limit conductance G(0). This illustrates that our new approach is promising for the direct assembly of CNT-based interconnects in integrated circuits (ICs) or other micro-electronic devices.
Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.
While somatic gene therapy has the potential to treat many genetic disorders, recent clinical trials suggest that an efficient and safe delivery vehicle for successful gene therapy is lacking. The current study examines the influence of two different preparation (the solvent evaporation method and the complex coacervation method) methods on the encapsulation of a model plasmid with chitosan. The ability of different molecular weights of chitosan to form nanoparticles with a plasmid, and particulated polymers to stabilize a plasmid in a supercoiled form, were examined by agarose gel electrophoresis. Protection of encapsulated pDNA offered by these nanoparticles from nuclease attack was confirmed by assessing degradation in the presence of DNase I, and the transformation of the plasmids with incubated nanoparticles were examined by beta-galactosidase assay. Model pDNA existed as a mixture of both supercoiled (84.2%) and open circular (15.8%) forms. Our results demonstrated that supercoiled forms decreased while open circular forms and fragmented linear forms increased during the preparation of formulations. F1 formulation prepared by the complex coacervation method protected the supercoiled form of pDNA effectively. There weren't any significant changes in nanoparticle size and zeta potential values at pH 5.5 for a period of 3 months, but differences in particle sizes were observed after lyophilization with a cryoprotective agent. The efficiency of nanoparticles mediated transformation to Escherichia coli cells was significantly higher than naked DNA or poly-L-lysine (PLL)-DNA polycation complexes. The transfection studies were performed in COS-7 cells. A 3-fold increase in gene expression was produced by nanoparticles as compared to the same amount of naked plasmid DNA (pDNA). These observations suggest that formulations with high molecular weight (HMW) chitosan can be an effective non-viral method of gene vector in animal studies.
Hydrogel nanoparticles have gained considerable attention in recent years as one of the most promising nanoparticulate drug delivery systems owing to their unique potentials via combining the characteristics of a hydrogel system (e.g., hydrophilicity and extremely high water content) with a nanoparticle (e.g., very small size). Several polymeric hydrogel nanoparticulate systems have been prepared and characterized in recent years, based on both natural and synthetic polymers, each with its own advantages and drawbacks. Among the natural polymers, chitosan and alginate have been studied extensively for preparation of hydrogel nanoparticles and from synthetic group, hydrogel nanoparticles based on poly (vinyl alcohol), poly (ethylene oxide), poly (ethyleneimine), poly (vinyl pyrrolidone), and poly-N-isopropylacrylamide have been reported with different characteristics and features with respect to drug delivery. Regardless of the type of polymer used, the release mechanism of the loaded agent from hydrogel nanoparticles is complex, while resulting from three main vectors, i.e., drug diffusion, hydrogel matrix swelling, and chemical reactivity of the drug/matrix. Several crosslinking methods have been used in the way to form the hydrogel matix structures, which can be classified in two major groups of chemically- and physically-induced crosslinking.
Chitosan and chitosan derivatives have been proposed as alternative and biocompatible cationic polymers for non-viral gene delivery. However, the low transfection efficiency and low specificity of chitosan is an aspect of this approach that must be addressed prior to any clinical applications. In the present study a chitosan derivative, galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine (Gal-PEG-CHI-g-PEI), was investigated as a potential hepatocyte-targeting gene carrier. The composition of Gal-PEG-CHI-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and the particle size and zeta potential of Gal-PEG-CHI-g-PEI/DNA complexes were measured using dynamic light scattering (DLS). The Gal-PEG-CHI-g-PEI exhibited lower cytotoxicity compared to PEI 25K as a control. Likewise, Gal-PEG-CHI-g-PEI/DNA complexes showed good hepatocyte specificity. Furthermore, Gal-PEG-CHI-g-PEI/DNA complexes transfected liver cells more effectively than PEI 25K in vivo after intravenous (i.v.) administration. Together, these results suggest that Gal-PEG-CHI-g-PEI, which has improved transfection efficiency and hepatocyte specificity both in vitro and in vivo, may be useful for gene therapy.
To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and (1)H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC 50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.
The lysozymic digestibility of partially N-acetylated chitosans was studied by measuring the reducing sugars produced and the molecular weights of their hydrolysates. Moderately N-deacetylated chitosans (MDC), obtained by N-deacetylation of chitin under heterogeneous conditions, were about four times more digestible at an early stage than partially N-acetylated chitosans (PAC-H) with similar acetyl content, prepared by N-acetylation of highly N-deacetylated chitosans under homogeneous conditions. The molecular weights of the hydrolysates of MDC decreased rapidly but gradually reached a constant value in contrast to the behaviour of PAC-H. The Km was 0.14 mM for 30% N-acetylated MDC and 0.12 mM for 65% N-acetylated PAC-H although the degree of N-acetylation of the latter was twice as much as the former. These differences were due to the different distribution patterns of N-acetyl groups in two types of the chitosans. MDC with 20-30% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues but PAC-H with about 30% acetyl content are random-type copolymers of N-acetyl-D-glucosamine and D-glucosamine units. PAC-H with more than 50% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues.
Nasal delivery is a potential alternative for systemic availability of drugs restricted to intravenous administration, such as peptide and protein drugs. Although nasal delivery avoids the hepatic first-pass effect, the enzymatic barrier of the nasal mucosa creates a pseudo-first-pass effect. The xenobiotic metabolic activity in the nasal epithelium has been investigated in several species including humans. The Phase I, cytochrome P-450 enzymes have been studied extensively for their toxicological significance, since these enzymes metabolize inhaled pollutants into reactive metabolites which may induce nasal tumors. The cytochrome P-450 activity in the olfactory region of the nasal epithelium is higher even than in the liver, mainly because of a three- to fourfold higher NADPH-cytochrome P-450 reductase content. Phase II activity has also been found in the nasal epithelium. The delivery of peptides and proteins has been hindered by the peptidase and protease activity in the nasal mucosa. The predominant enzyme appears to be aminopeptidase among other exopeptidases and endopeptidases. The absorption of peptide drugs can be improved by using aminoboronic acid derivatives, amastatin, and other enzyme inhibitors as absorption enhancers. It is possible that some of the surfactants, e.g., bile salts, increase absorption by inhibiting the proteolytic enzymes. Thus, in addition to the permeation barriers, there also exists an enzymatic barrier to nasal drug delivery, which is created by metabolic enzymes in the nasal epithelium.
To identify the moiety responsible for nuclear localization of the SV40 structural protein Vp3 in its natural environment, a transfection vector containing the entire coding regions of Vp2, Vp3, and agnoprotein, and one-third of the coding region of Vp1, was constructed. Several mutations were introduced into the plasmid and the subcellular distribution of Vp3 or mutant Vp3 was examined following DEAE-dextran-mediated DNA transfection into TC7 cells. Our study shows that Vp3 is synthesized and is transported into the nucleus in the absence of Vp2, agnoprotein, and intact Vp1. However, in the absence of its carboxyl-terminal 35 amino acids, the truncated Vp3 is limited to a cytoplasmic and perinuclear accumulation. Thus, the carboxyl 35 amino acids of Vp3 are required for its nuclear localization and may contain a nuclear accumulation signal.
The karyophilic protein N1 (590 amino acids) is an abundant soluble protein of the nuclei of Xenopus laevis oocytes where it forms defined complexes with histones H3 and H4. The amino acid sequence of this protein, as deduced from the cDNA, reveals a putative nuclear targeting signal as well as two acidic domains which are candidates for the interaction with histones. Using two different histone binding assays in vitro we have found that the deletion of the larger acidic domain reduces histone binding drastically to a residual value of approximately 15% of the complete molecule, whereas removal of the smaller acidic domain only slightly reduces histone complex formation in solution, but infers more effectively with binding to immobilized histones. In the primary structure of the protein both histone-binding domains are distant from the conspicuous nuclear accumulation signal sequence (residues 531-537) close to the carboxy terminus which is very similar to the SV40 large T-antigen nuclear targeting sequence. Using a series of N1 mutants altered by deletions or point mutations we show that this signal is required but not sufficient for nuclear accumulation of protein N1. The presence of an additional, more distantly related signal sequence in position 544-554 is also needed to achieve a level of nuclear uptake equivalent to that of the wild-type protein. Results obtained with point mutations support the concept of two nuclear targeting sequences and emphasize the importance of specific lysine and arginine residues in these signal sequences.
The presumed open reading frame for mouse FGF3, starting at the most 5' AUG codon, predicts a hydrophobic N-terminus characteristic of a signal peptide for secretion. However, in reticulocyte lysates and transfected COS-1 cells, the full-length Fgf-3 cDNA is translated almost exclusively from an upstream CUG codon. The resultant products are distributed in both the nucleus and the secretory pathway, implying that the single CUG-initiated form of FGF3 has dual fates. By analysing a series of deletion and replacement mutants and by linking parts of FGF3 to a heterologous protein, we show that secretion is mediated by cleavage adjacent to the previously defined signal peptide, whereas nuclear localization is determined primarily by a classical but relatively weak bipartite motif. In the context of FGF3, nuclear localization also requires the N-terminal sequences which lie upstream of the signal peptide. Thus, the subcellular fate of FGF3 is determined by the competing effects of signals for secretion and nuclear localization within the same protein, rather than by alternative initiation or processing.
Chitosan was selectively N-acylated with various carboxylic anhydrides, e.g., acetic, propionic, n-butyric, n-valeric and n-hexanoic anhydrides, in the presence of methanol. The degree of N-acylation of about 20-50% was obtainable without occurrence of gelation by using carboxylic anhydrides of 0.3-1.2 mol per glucosamine residue. In vitro blood compatibility tests of N-acyl chitosans were performed by rheological measurement, blood clotting test and scanning electron microscopic observation for human blood and plasma protein. The rheological measurement of coagulation of plasma protein, considering the shear flow effect of blood, gave precise and quantitative results compared with other methods. N-Acyl chitosans showed more blood compatible properties than N-acetyl chitosan and, in particular, N-hexanoyl chitosan was the most compatible. Enzymatic degradation was also investigated by adding a lysozyme solution to the N-acyl chitosan solution and film, incubating at 37 degrees C. N-Acyl chitosans had as high a susceptibility to lysozyme as N-acetyl chitosans. It was considered that the amount of derivatized groups and the physical form of N-acyl chitosans contributed to biodegradability. The molecular weight (Mw) of the material liberated from the N-acyl chitosan film by the action of lysozyme was 2 x 10(4) - 10 x 10(4).
We have designed a cationic amphipathic peptide, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), that binds to DNA, destabilizes membranes, and mediates DNA transfection. KALA undergoes a pH-dependent random coil to amphipathic alpha-helical conformational change as the pH is increased from 5.0 to 7.5. One face displays hydrophobic leucine residues, and the opposite face displays hydrophilic lysine residues. KALA-mediated release of entrapped aqueous contents from neutral and negatively charged liposomes increases with increasing helical content. KALA binds to oligonucleotides or plasmid DNA and retards their migration in gel electrophoresis. It displaces 50% of ethidium bromide from DNA at a charge ratio (+/-) of 0.9/1. In cultured cells, KALA assists oligonucleotide nuclear delivery when complexes are prepared at a 10/1 (+/-) charge ratio. KALA/DNA (10/1)(+/-) complexes mediate transfection of a variety of cell lines. The KALA sequence provides a starting point for a family of peptides that incorporate other functions to improve DNA delivery systems.
Targeting gene therapy vectors to abundant receptors on airway epithelia may allow a significant enhancement of gene delivery and thereby be of particular importance for the gene therapy of cystic fibrosis. Alpha9beta1-integrins are highly expressed throughout the human airway epithelia in vivo, irrespective of any particular clinical status. Aiming to improve the targeting of our non-viral integrin-mediated gene transfer systems to airway epithelia, we searched for a short tenascin C-derived peptide which would bind to these integrins. By utilizing recombinant bacteriophages that display overlapping regions of the third fibronectin type III repeat of tenascin C (TNfn3), we were able to localize its alpha9beta1-integrin binding site to the B-C loop of TNfn3. A synthetic Pro-Leu-Ala-Glu-Ile-Asp-Gly-Ile-Glu-Leu-Thr-Tyr peptide (PLAEIDGIELTY) was shown to displace alpha9beta1-integrin-expressing cells completely from binding to TNfn3. This peptide, therefore, may prove useful both for the examination of the functional importance of alpha9beta1-integrins in vivo and the development of gene therapy vectors or drugs targeting these integrins.
Recent studies in our laboratory have shown that chitosan, a polycationic polymer of glucosamine, can facilitate the transfection of HeLa cells with a plasmid that codes for beta-galactosidase. Although chitosan can bind to DNA and other polyanions, the kinetics of complexation might differ depending on the polyanion tested. This evidence suggests that, in addition to ionic interactions, the carbohydrate backbone of chitosan might have an important role in the process of transfection. Beads prepared by the complexation of chitosan with polyphosphate were used to investigate the nature of cellular interactions with chitosan. HeLa cells bound to chitosan-polyphosphate beads could be readily displaced from the beads with methyl alpha-D-mannopyranoside but not with NaCl. Membrane proteins solubilized by CHAPS bound readily to chitosan-polyphosphate beads. A major fraction of the membrane proteins could be eluted from the beads with methyl alpha-D-mannopyranoside. These results suggest that non-ionic interactions between the carbohydrate backbone of chitosan and cell surface proteins might have an important role in the chitosan-mediated transfection of HeLa cells.
The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1 +/- 0.03%; mean +/- SD, n = 3) based on free chitosan as 1.0 was capable of binding 1.4 +/- 0.1 mM calcium, 2.0 +/- 0.1 mM zinc and 1.9 +/- 0.03 mM cobalt (mean +/- SD, n = 3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan-EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan-EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.
We investigated the transport- and metabolism properties of three peptides in monolayers of human nasal epithelial cells. The effective permeability coefficients of thyrotropin-releasing hormone, met-enkephalin and human recombinant insulin were found to be 4.5, 4.4 and 0.4 x 10(-7) cm/s, respectively. The permeability was inversely proportional to the molecular weight and one order of magnitude lower than in excised nasal mucosa of rabbits. The metabolic cleavage of thyrotropin-releasing hormone (TRH) to the free acid by cytosolic prolyl-endopeptidase was also detected in human nasal cell monolayers, suggesting that ca. 10% of the total amount of TRH is transported via a transcellular pathway. Met-enkephalin is a substrate for aminopeptidases, located on the apical membrane of nasal epithelial cells. Metabolites and enzyme activity are comparable with literature data. Our studies demonstrate that not only morphological, but also functional properties of human nasal epithelial cells are preserved under in vitro conditions. Such a cell culture model based on human nasal cells could be beneficial for the characterization of peptide transport on a cellular level and for investigation of the absorption enhancer mechanism. Further studies are necessary, however, to establish correlations between in vitro permeabilities in cell cultures and nasal drug absorption in animals and humans.
Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed.
Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene.
In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here.
Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.
Currently in vivo gene delivery by synthetic vectors is hindered by the limited diffusibility of complexes in extra-cellular fluids and matrices. Here we show that certain formulations of plasmid DNA with linear polyethylenimine (22 kDa PEI, ExGene 500) can produce complexes that are sufficiently small and stable in physiological fluids so as to provide high diffusibility. When plasmid DNA was formulated with 22 kDa PEI in 5% glucose, it produced a homogeneous population of complexes with mean diameters ranging from 30 to 100 nm according to the amount of PEI used. In contrast, formulation in physiological saline produced complexes an order of magnitude greater (> or = 1 micron). Intraventricular injection of complexes formulated in glu-cose showed the complexes to be highly diffusible in the cerebrospinal fluid of newborn and adult mice, diffusing from a single site of injection throughout the entire brain ventricular spaces. Transfection efficiency was followed by histochemistry of beta-galactosidase activity and double immunocytochemistry was used to identify the cells transfected. Transgene expression was found in both neurons and glia adjacent to ventricular spaces. Thus, this method of formulation is promising for in vivo work and may well be adaptable to other vectors and physiological models.
Efficient DNA delivery is a prerequisite for the successful implementation of molecular antiviral strategies against chronic viral hepatitis and gene therapy in general. The cationic polymer polyethylenimine (PEI) has recently been explored as a gene transfer vector in various cell types in vitro and in vivo. In this study, we evaluated a linear PEI derivative (lPEI) as a vector for gene and oligodeoxynucleotide transfer into hepatocytes in vitro and in vivo. A simple protocol was developed that allowed transfection of up to 50% of primary hepatocytes in vitro. In addition, fluorescent oligodeoxynucleotides were efficiently delivered to the liver in vivo after intravenous injection into Pekin ducks. Thus, lPEI mediates highly efficient gene and oligodeoxynucleotide transfer into primary hepatocytes and is potentially useful for DNA delivery in vivo.