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Elucidating the Molecular Interactions of Encapsulated Doxorubicin Within a Non-ionic, Thermoresponsive Polyester Coacervate
Abstract
Thermoresponsive polymers that display a lower critical solution temperature (LCST), are attractive drug delivery systems (DDS) due to their potential to encapsulate and release therapeutics in a sustained manner as a function of temperature input. To attain the full potential of such DDS, methods that illustrate the details of drug-polymer interactions are necessary. Here, we synthesized a non-ionic, coacervate-forming, thermoresponsive polyester to encapsulate doxorubicin (Dox) and used solution state NMR spectroscopy and fluorescence microscopy techniques to probe the interactions between the polymer and Dox at the molecular level. The incomplete dehydration provides a matrix for encapsulation of sensitive therapeutics and preserving their activity, while the low hysteresis properties of the polyester provides rapid transition from soluble to coacervate phase. Saturation Transfer Difference (STD) NMR revealed the Dox-polymer interactions within the coacervates. 1H-1H Nuclear Overhauser Effect Spectroscopy (NOESY) cross-peak differences of Dox confirmed the Dox-polymer interactions. Diffusion-Ordered Spectroscopy (DOSY) revealed the slower diffusion rate of Dox in the presence of polyester coacervates. These studies illustrate how the state of the polyester (below and above LCST) affects the polyester-Dox interactions and offers details of the specific functional groups involved in these interactions. Our results provide a framework for future investigations aimed at characterizing fundamental interactions in polymer-based DDS.
Supplementary resource (1)
Data
July 2020
... Reproduced with permission from Macromolecules, 2020, 53, 5465. Copyright 2020 American Chemical Society.Joy et al. have worked on different coacervates based on biodegradable, thermoresponsive polyester polyelectrolytes, and they have used such coacervates for the encapsulation of different hydrophobic materials[106][107][108][109]. ...
Individual amphiphiles, polymers, and colloidal dispersions influenced by temperature, pH, and environmental conditions or interactions between their oppositely charged pairs in solvent medium often produce solvent-rich and solvent-poor phases in the system. The solvent-poor denser phase found either on the top or the bottom of the system is called coacervate. Coacervates have immense applications in various technological fields. This review comprises a concise introduction, focusing on the types of coacervates, and the influence of different factors in their formation, structures, and stability. In addition, their physicochemical properties, thermodynamics of formation, and uses and multifarious applications are also concisely presented and discussed.
Aqueous two-phase systems (ATPS) are useful in various applications, from purification and separation of biomolecules to wastewater treatment. While they have great utility on their own, there is great interest in discovering how their emulsions, comprising droplets of one aqueous phase dispersed in the other aqueous phase, might be stabilized to enhance their functionality and applications. There are several examples of these systems, but the two most common systems found in the literature are PEG–dextran and complex coacervate ATPS. In this Review, we discuss these systems, their utility, and many different approaches for stabilizing their water/water (w/w) emulsions. We highlight examples wherein interfacial stabilizers such as liposomes, polymers of diverse architectures, colloids of varied shapes and morphologies, and even whole cells have been employed. These stabilization approaches for both PEG–dextran and complex coacervate ATPS are discussed. We conclude with a discussion of the applications of these ATPS and how they can benefit from the creation of corresponding w/w emulsions with stabilized droplets.
Conjugation of maltopentaose to water-soluble homo-poly(sarcosine) induced self-association and formed nanospheres (-150nm) in water although homo-poly(sarcosine) was water-soluble and did not form any aggregates. Fluorosecnt probes experiments showed that the...
Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins' sequence-structure-function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.
Known for their adaptability to surroundings, capability of transport control of molecules, or the ability of converting one type of energy to another as a result of external or internal stimuli, responsive polymers play a significant role in advancing scientific discoveries that may lead to an array of diverge applications. This review outlines recent advances in the developments of selected commodity polymers equipped with stimuli‐responsiveness to temperature, pH, ionic strength, enzyme or glucose levels, carbon dioxide, water, redox agents, electromagnetic radiation, or electric and magnetic fields. Utilized diverse applications ranging from drug delivery to biosensing, dynamic structural components to color‐changing coatings, this review focuses on commodity acrylics, epoxies, esters, carbonates, urethanes, and siloxane‐based polymers containing responsive elements built into their architecture. In the context of stimuli‐responsive chemistries, current technological advances as well as a critical outline of future opportunities and applications are also tackled. This review outlines recent advances in the developments of commodity polymers capable of stimuli‐responsiveness to temperature, pH, ionic strength, enzyme/glucose levels, CO2, H2O, redox agents, electromagnetic radiation, or electric/magnetic fields. Diverse applications ranging from drug delivery to biosensing, dynamic structural components to color‐changing coatings in acrylics, epoxies, esters, carbonates, urethanes, and siloxane‐based polymers containing responsive elements are outlined.
Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell—a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated “membraneless organelles” that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid–liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein‐containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted.
Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-β3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-β3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. Statement of Significance: This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.
Presented here is a novel method for encapsulating proteins into biodegradable, thermoresponsive coacervate-type polyesters. Bovine serum albumin (BSA) was efficiently incorporated into coacervate droplets via a simple thermoresponsive encapsulation mechanism. Tunable modular systems for encapsulation such as the one presented here may be useful in a range of protein delivery applications.
H, ¹H-¹H COSY and ¹³C NMR techniques were proven as effective methods for the analysis of functionalized doxorubicin (DOX) and daunorubicin (DAU) derivatives. These compounds represent important drug conjugate intermediates that can be coupled to a variety of nanocarriers. NMR spectroscopy was shown to be an efficient method for following the progress of drug modification and can also be useful for evaluation of the functionalized structures purity. Additionally, a correction of the chemical shifts reported in the literature for the relevant drugs e.g. DOX⋅HCl and DAU⋅HCl is presented.
Selective hits for the glutaredoxin ortholog of Brucella melitensis are determined using STD NMR and verified by trNOE and (15)N-HSQC titration. The most promising hit, RK207, was docked into the target molecule using a scoring function to compare simulated poses to experimental data. After elucidating possible poses, the hit was further optimized into the lead compound by extension with an electrophilic acrylamide warhead. We believe that focusing on selectivity in this early stage of drug discovery will limit cross-reactivity that might occur with the human ortholog as the lead compound is optimized. Kinetics studies revealed that lead compound 5 modified with an ester group results in higher reactivity than an acrylamide control; however, after modification this compound shows little selectivity for bacterial protein versus the human ortholog. In contrast, hydrolysis of compound 5 to the acid form results in a decrease in the activity of the compound. Together these results suggest that more optimization is warranted for this simple chemical scaffold, and opens the door for discovery of drugs targeted against glutaredoxin proteins-a heretofore untapped reservoir for antibiotic agents.
We report on a new class of thermoresponsive biodegradable polyesters (TR-PE) inspired by polyacrylamides and elastin-like proteins (ELPs). The polyesters display reversible phase transition with tunable cloud point temperatures (Tcp) in aqueous solution as evidenced by UV-vis spectroscopy, 1H NMR, and DLS measurements. These polyesters form coacervate droplets above their lower critical solution temperature (LCST). The Tcp of the polyesters is influenced by the solutes such as urea, SDS, and NaCl. The Tcp of the copolymers shows a linear correlation with the composition of the polyesters indicating the ability to tune the phase change temperature. We also show that such thermoresponsive coacervates are capable of encapsulating small molecules such as Nile Red. Furthermore, the polyesters are hydrolytically degradable.
By choosing poly(N,N-diethylacrylamide) which lacks the possibility to form intra- or inter-chain hydrogen bonds, we studied its chain association and dissociation in water by using laser light scattering (LLS), ultrasensitive differential scanning calorimetry (US-DSC) and Fourier transform infrared spectroscopy (FTIR). As the solution temperature increases, the average hydrodynamic radius (R(h)) and average radius of gyration (R(g)) decrease, indicating the intrachain shrinking. When the temperature is higher than its lower critical solution temperature (LCST, approximately 30 degrees C), the apparent weight-average molar mass (M(w,app)) increases, reflecting the interchain association. At the same time, FTIR study reveals that as the temperature increases, the area ratio of two absorption peaks, respectively, associated to one hydrogen bonded carbonyl >C=O...H-O-H and free carbonyl >C=O groups increases, while that related to two hydrated hydrogen bonded carbonyl groups decreases, indicating heating-induced dehydration. In the reversible cooling process, R(h), R(g), M(w,app) and area ratios of the absorption peak are similar to those in the heating process for each given temperature, indicating that there is no hysteresis in the interchain association and dissociation. This present study confirms that the hysteresis previously observed for a sister polymer, poly(N-isopropylacrylamide), is due to the formation of some additional hydrogen bonds in its collapsed state at temperatures higher than the LCST.
The indications for pegylated liposomal doxorubicin (doxil) are expanding. We, therefore, wished to assess the safety of delivering doses exceeding 500 mg/m2 of doxil to patients with solid tumors.
Subjects accrued to eight phase I and II protocol studies conducted at two institutions, were assessed for cardiac function at baseline and at specified intervals by MUGA scans. In this retrospective analysis, the findings of 42 patients, from the total of 237 entered, who had reached or exceeded cumulative doses of 500 mg/m2 (range 500-1500 mg/m2) were reviewed. Changes in left ventricular ejection fraction (LVEF), and in clinical cardiac status were analyzed. Six patients, three who had received prior doxorubicin, also underwent endomyocardial biopsies after cumulative doses of 490-1320 mg/m2.
None of the 42 patients had clinical congestive heart failure (CHF) secondary to cardiomyopathy. Post doxil MUGA scans were available for 41 of the 42 patients. Five had a drop of 10% or more in LVEF; three of these had received prior doxorubicin. Billingham endomyocardial biopsy scores ranged from 0-1 in five patients, while the sixth had a score of 1.5 after both 900 mg/m2 and 1320 mg/m2 doxil. Of a remaining 195 patients, 1 episode of CHF was recorded in a patient who had received 312 mg/m2 doxil over 120 mg/m2 of mitoxantrone and chest radiation.
Cumulative doses in excess of 500 mg/m2 of doxil appear to carry a considerably lesser risk of cardiomyopathy as judged by serial LVEF's and clinical follow-up, than is generally associated with free doxorubicin. Heart biopsies have provided reassuring data in a small number of patients, even if pretreated with doxorubicin. However, since three doxorubicin pretreated patients were among the five experiencing drops in LVEF, more data are warranted on such patients.
Advances in biomaterials for drug delivery are enabling significant progress in biology and medicine. Multidisciplinary collaborations between physical scientists, engineers, biologists, and clinicians generate innovative strategies and materials to treat a range of diseases. Specifically, recent advances include major breakthroughs in materials for cancer immunotherapy, autoimmune diseases, and genome editing. Here, strategies for the design and implementation of biomaterials for drug delivery are reviewed. A brief history of the biomaterials field is first established, and then commentary on RNA delivery, responsive materials development, and immunomodulation are provided. Current challenges associated with these areas as well as opportunities to address long‐standing problems in biology and medicine are discussed throughout.
In this article, we have unveiled the aggregation behavior of a potent chemotherapeutic drug, doxorubicin hydrochloride (Dox) in a well known imidazolium based surface active ionic liquid (SAIL), 1-octyl-3-methylimidazolium chloride (C8mimCl). The aggregates formed by Dox in C8mimCl have been characterized using dynamic light scattering (DLS), fluorescence lifetime imaging microscopy (FLIM), high resolution transmission electron microscopy (HR-TEM), analytical transmission electron microscopy (analytical TEM), field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and fourier-transform infrared spectroscopy (FTIR) measurements. It is found that Dox forms large spherical aggregates in presence of C8mimCl SAIL. We have also explored the driving force behind this aggregation behavior of Dox in C8mimCl. Furthermore, it is observed that in presence of a common bile salt, sodium cholate (NaCh), Dox/ C8mimCl spherical aggregates disrupt to form rod like fibrillar aggregates. Therefore, formation of spherical aggregates and also its disruption into rod like fibrillar aggregates have been performed and this is expected to open a new scope for the design of a new generation smart drug delivery system where drug itself aggregates to form the delivery system.
Two poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PND) statistical copolymers and a series of three poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-polystyrene (PND-b-PS-C12) diblock polymers were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization, in which the molecular weight of the thermoresponsive PND corona block was held constant while the polystyrene core block length was varied. The corona thickness and density of the micelles in phosphate-buffered saline (PBS, pH = 6.5) were quantified by a combination of dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). Two hydrophobic model drugs, phenytoin and nilutamide, were used to examine the drug–polymer interactions in aqueous solution. Intermolecular interactions between the diblock polymer micelle corona and both drugs were revealed by 2D ¹H nuclear Overhauser effect spectroscopy (NOESY). The drug–polymer “binding” strength, quantified by diffusion ordered NMR spectroscopy (DOSY), increased as corona density of the diblock polymer micelle increased for both drugs. The in vitro dissolution of the amorphous solid dispersions was systematically investigated as a function of drug type, drug loading, and the solution-state assembly of the polymers by using either a selective or nonselective spray drying solvent. Forming micelles prior to spray drying significantly enhanced phenytoin dissolution and supersaturation maintenance for the diblock polymers by storing the drug molecules in the corona.
Diffusion ordered spectroscopy (DOSY) is a well established NMR method that reports diffusion coefficients for individual resonances in NMR spectra. DOSY is primarily used to analyse mixtures of small molecules and the oligomeric state of biomolecules. DOSY has also been used to analyse polymers and investigate micellization properties but different acquisition and processing parameters are recommended for polymers. In particular, the molecular weight dispersion of polymers and micelles are at odds with the physical limits of DOSY. Confidence in the quality of published DOSY data is lowered when critical parameters are poorly optimized or not reported. This tutorial provides a ‘top ten’ of DOSY parameters, an explanation of their source and importance, as well as suggested starting parameters and optimization for polymer/micelle samples. By following these guidelines, DOSY can emerge from being an occasional method to confirm data obtained from other experimental techniques, to one that provides strong physical evidence in its own right as the originators of DOSY intended.
Poly(N-acryloylsarcosine methyl ester) (PNASME), a thermoresponsive N-ester substituted polyacrylamide has well-defined molecular weight and low Ð, was synthesized by solution RAFT polymerization. The synthesized PNASME exhibited a cloud point temperature (Tcp) or lower critical solution temperature (LCST) at 44.0 oC. It was found that various factors could affect Tcp of PNASME, e.g, the polymer molecular weight, the polymer concentration, the salt effect on Tcp following the order of Hofmeister series, urea increasing Tcp, and phenol decreasing Tcp, respectively. The mechanism of urea and phenol inducing the phase transition of PNASME was revealed by 2D NOESY spectra, and how the hydrogen bonding between PNASME and the solutes of urea and phenol affected the phase transition of PNASME was clarified out. The contrary effect on the Tcp of PNASME induced by urea and phenol affords the convenience to adjust the thermoresponse of PNASME, and the tunable thermoresponse of the diblock copolymer of poly(N-acryloylsarcosine methyl ester)-block-poly(N-isopropylacrylamide) is demonstrated. The present study is believed to offer a new pathway to tune the solution behavior of thermoresponsive polymers.
The focus of this review is on the class of transiently thermoresponsive polymers. These polymers are thermoresponsive, but gradually lose this property upon chemical transformation - often a hydrolysis reaction - in the polymer side chain or backbone. An overview of the different approaches used for the design of these polymers along with their physicochemical properties is given. Their amphiphilic properties and degradability into fully soluble compounds make this class of responsive polymers attractive for drug delivery and tissue engineering applications. Examples of these are also provided in this review.
Thermoresponsive polymers that undergo reversible phase transition by responding to an environmental temperature change, in particular polymers showing lower critical solution temperature (LCST), are frequently used as smart materials that have found increasing applications. Recently, there has been a rapid growth in interest on LCST polymers and many new research groups are entering the field from a wide range of application areas. While it is great to see more researchers working on LCST polymers, the downside of this rapid growth is that the fundamentals of the LCST phase transition behavior are not always clearly known and respected. Hence, this focus article provides a systematic discussion of the key aspects of the LCST behavior of polymers starting from fundamentals of LCST behavior to practical determination of cloud point temperature (T-cp). Finally, we offer a basic set of recommended measuring conditions for determination of T-cp (10 mg mL(-1); 0.5 degrees C min(-1); 600 nm) to facilitate the comparison of the LCST behavior and T-cp values of polymers developed and studied in different laboratories around the globe, which is nowadays nearly impossible since various techniques and parameters are being utilized for the measurements. It should be noted that these recommended conditions serve as a robust tool for turbidimetry, which is one out of the many characterization techniques one should utilize to fully understand LCST behavior of polymers.
‘Smart’ bioresponsive materials that are sensitive to biological signals or to pathological abnormalities, and interact with or are actuated by them, are appealing therapeutic platforms for the development of next-generation precision medications. Armed with a better understanding of various biologically responsive mechanisms, researchers have made innovations in the areas of materials chemistry, biomolecular engineering, pharmaceutical science, and micro- and nanofabrication to develop bioresponsive materials for a range of applications, including controlled drug delivery, diagnostics, tissue engineering and biomedical devices. This Review highlights recent advances in the design of smart materials capable of responding to the physiological environment, to biomarkers and to biological particulates. Key design principles, challenges and future directions, including clinical translation, of bioresponsive materials are also discussed.
Recently, we reported a new class of biodegradable, thermoresponsive polyesters (TR-PEs) inspired by polyacrylamides and elastin-like peptides (ELPs). The polyesters exhibit tunable cloud point temperatures (Tcp) and thermoresponsive coacervation in aqueous solution as shown via UV-vis spectroscopy, 1H NMR, and DLS. However, the Tcp of all TR-PEs remained low (<15 °C), and higher thermoresponsivity would be beneficial for many applications. This study examines the synthesis, polymerization, and analysis of a new monomer bearing a more hydrophilic pendant group, bis-2-methoxyethylamine (bMoEtA). The resulting TR-PE, TR-bMoEtAPE, displays a threefold increase in Tcp (ca. 50 °C) that is affected by solution (DI water vs. phosphate buffered saline), concentration (1–40 mg mL−1) molecular weight (20–130 kDa), and cosolutes (Hofmeister salts and urea). The Tcp and Tg of random TR-bMoEtAPE copolymers can be tuned via comonomer feed. Variable temperature 1H NMR indicated a cooperative coacervation mechanism above Tcp, further reinforced by DLS measurements. As evidenced by UV-vis and SEC analysis, TR-bMoEtAPE underwent rapid degradation over a period of 7 days in DI water and PBS. Finally, cytotoxicity studies suggested that TR-bMoEtAPE is non-cytotoxic even at high concentrations (ca. 1000 μg mL−1). The increased Tcp and tunability suggests TR-bMoEtAPE as a potential candidate for future functionalized TR-PE therapeutic-delivery systems.
Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.
Devices resident in the stomach-used for a variety of clinical applications including nutritional modulation for bariatrics, ingestible electronics for diagnosis and monitoring, and gastric-retentive dosage forms for prolonged drug delivery-typically incorporate elastic polymers to compress the devices during delivery through the oesophagus and other narrow orifices in the digestive system. However, in the event of accidental device fracture or migration, the non-degradable nature of these materials risks intestinal obstruction. Here, we show that an elastic, pH-responsive supramolecular gel remains stable and elastic in the acidic environment of the stomach but can be dissolved in the neutral-pH environment of the small and large intestines. In a large animal model, prototype devices with these materials as the key component demonstrated prolonged gastric retention and safe passage. These enteric elastomers should increase the safety profile for a wide range of gastric-retentive devices.
The search of targeted drug delivery systems requires the design of drug-carrier complexes, which could both reach the malignant cells and preserve the therapeutic substance activity. A promising strategy aimed at enhancing the uptake and reducing the systemic toxicity is to bind covalently the drug to a cell-penetrating peptide. For understanding the structure-activity relation in such preparations, the chemotherapeutic drug doxorubicin was investigated by unrestrained molecular dynamics simulations, supported by NMR, which yielded its molecular geometry in aqueous environment. Furthermore, the structure and dynamics of a conjugate of the drug with a cell-penetrating peptide was obtained from molecular dynamics simulations in aqueous solution. The geometries of the unbound compounds were characterized at different temperatures, the extent to which they change after covalent binding and whether/how they influence each other into the drug-peptide conjugate. The main structural fragments that affect the conformational ensemble of every molecule were found. The results show that the transitions between different substructures of the three compounds require modest amount of energy. At increased temperature either more conformations become populated as a result of the thermal fluctuations, or the relative shares of the various conformers equalize at the nanosecond scale. These frequent structural interconversions suggest expressed conformational freedom of the molecules. Conjugation into the drug-peptide compound partially immobilizes the molecules of the parent compounds. Nevertheless, flexibility still exists as well as an effective intra- and intermolecular hydrogen bonding that stabilizes the structures. We observe compact packing of the drug within the peptide that is also based on stacking interactions. All this outlines the drug-peptide conjugate as a prospective building block of a more complex drug-carrier system.
Proteins have gained increasing success as therapeutic agents; however, challenges exist in effective and efficient delivery. In this work, we present a simple and versatile method for encapsulating proteins via complex coacervation with oppositely charged polypeptides, poly(l-lysine) (PLys) and poly(d/l-glutamic acid) (PGlu). A model protein system, bovine serum albumin (BSA), was incorporated efficiently into coacervate droplets via electrostatic interaction up to a maximum loading of one BSA per PLys/PGlu pair and could be released under conditions of decreasing pH. Additionally, encapsulation within complex coacervates did not alter the secondary structure of the protein. Lastly the complex coacervate system was shown to be biocompatible and interact well with cells in vitro. A simple, modular system for encapsulation such as the one presented here may be useful in a range of drug delivery applications.
The ability to efficiently deliver a drug to a tumor site is dependent on a wide range of physiologically imposed design constraints. Nanotechnology provides the possibility of creating delivery vehicles where these design constraints can be decoupled, allowing new approaches for reducing the unwanted side effects of systemic delivery, increasing targeting efficiency and efficacy. Here we review the design strategies of the two FDA-approved anti-body–drug conjugates (Brentuximab vedotin and Trastuzumab emtansine) and the four FDA-approved nanoparticle-based drug delivery platforms (Doxil, DaunoXome, Marqibo, and Abraxane) in the context of the challenges associated with systemic targeted delivery of a drug to a solid tumor. The lessons learned from these nanomedicines provide an important insight into the key challenges associated with the development of new platforms for systemic delivery of anti-cancer drugs.
The conformation of adriamycin has been investigated by proton NMR spectroscopy at 500 MHz in D2O and DMSO. Two-dimensional phase-sensitive COSY spectra are recorded to obtain three-bond and long-range coupling constants and hence torsional angles. Interproton distances are estimated from 2D phase-sensitive NOESY spectra using r(1H–2H) = 2.36 Å as internal reference. Pairs of protons correlated by spin-spin coupling give NOE cross peaks with interproton distances < 3.3 Å. Besides these contacts, several other NOE contacts are observed between protons within ring A and within the daunosamine sugar, and between ring A and the daunosamine sugar. It is also observed that protons 7H, 8axH and 8eqH of ring A are close to protons 1′H, 3′H, 4′H, 5′H and 5′CH3 of the daunosamine sugar with distances lying in the range 2.40–3.90 Å. The 6OH proton is in the proximity of the 1′H and 3′NH+3 protons of the daunosamine sugar. The NOE contacts 5t'H8eqH, 5′H8eqH, 5′CH38axH, 5′CH38eqH and 1′H7H are found to have a strong influence on the solution conformation of the adriamycin molecule determined on the basis of coupling constants and interproton distances.
The conformational properties of daunomycin (1) and several analogues (2)–(16) have been investigated by 1H n.m.r. in different solvents, CDCI3, D2O, DMSO, dioxane, and pyridine. From H,H three-bond and long-range coupling constants, the geometry of ring A in each solvent has been determined, through a Karplus–Altona equation, which includes a correction for the substiuent electronegativity. Conformers 9H8, 8H9, 9S, S9, and S8 have been found in solution, and in several cases, especially in DMSO, more than one conformer is present at equilibrium. The equilibrium is always fast compared with n.m.r. times and the relative populations of each conformer have been calculated from experimental and model coupling constants by using a least-squares procedure. Two factors have been recognized to be responsible for the shape of ring A: the intramolecular hydrogen-bond 9–OH O(7) and steric interactions between peri substituents on the A and B rings. Evidence for this hydrogen-bonding has been obtained by dilution experiments, and, for the peri interactions, by evaluating the substituent effects on the conformational preference. Other intramolecular hydrogen bonds have been proved not to exist in solution. The influence of the solvents on the shape of ring A has also been studied. All the results are discussed and compared with those obtained in the solid phase. Daunosamine moiety has also been analysed, and the conformation of the sugar ring is always 1′C4. (L) in all the solvents examined.
Biodegradable poly(dl-lactic acid) nanoparticles (NP) produced by the salting-out process were evaluated in vitro and in vivo for their sustained release properties and their capacity to temporarily avoid the mononuclear phagocyte system (MPS). In vivo, NP loaded with neuroleptic compound savoxepine were able to provide sustained plasma levels after intramuscular and intravenous injection. Intramuscularly injected NP remained at the site of injection, whereas intravenously injected NP were located mostly in the MPS. The NP were successfully coated during the preparation procedure with PEG 6000 and PEG 20 000. In vitro, these coatings provided a protective barrier against extensive uptake by human monocytes, at least in plasma. Analysis of plasma proteins adsorbed on NP and in vitro experiments on isolated cells revealed some differences between the opsonization process of plain and coated NP. Plain and PEG 20000-coated NP loaded with photosensitizer hexafluoro zinc phthalocyanine were injected intravenously to mice bearing EMT-6 mouse mammary tumors. The protective coating produced a dramatic increase of the photosensitizer concentration in blood and tumor as well as a decrease in the MPS sequestration. These findings suggest that surface-modified NP should prove useful for the delivery of chemotherapeutic drugs to tumoral tissues.
Interest in thermoresponsive polymers has steadily grown over many decades, and a great deal of work has been dedicated to developing temperature sensitive macromolecules that can be crafted into new smart materials. However, the overwhelming majority of previously reported temperature-responsive polymers are based on poly(N-isopropylacrylamide) (PNIPAM), despite the fact that a wide range of other thermoresponsive polymers have demonstrated similar promise for the preparation of adaptive materials. Herein, we aim to highlight recent results that involve thermoresponsive systems that have not yet been as fully considered. Many of these (co)polymers represent clear opportunities for advancements in emerging biomedical and materials fields due to their increased biocompatibility and tuneable response. By highlighting recent examples of newly developed thermoresponsive polymer systems, we hope to promote the development of new generations of smart materials.
To address the practical issues of polymer molecular weight determination, the first accurate polymer weight-average molecular weight determination method in diverse living/controlled polymerization via DOSY (diffusion-ordered NMR spectroscopy) is reported. Based on the linear correlation between the logarithm of diffusion coefficient (log D) and the molecular weights (log Mw), external calibration curves were created to give predictions of molecular weights of narrowly-dispersed polymers. This method was successfully applied to atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), and ring-opening metathesis polymerization (ROMP), with weight-average molecular weights given by this method closely correlated to those obtained from GPC measurement.
This review focuses on polymers with upper critical solution temperature (UCST) in water or electrolyte solution and provides a detailed survey of the yet few existing examples. A guide for synthetic chemists for the design of novel UCST polymers is presented and possible handles to tune the phase transition temperature, sharpness of transition, hysteresis, and effectiveness of phase separation are discussed. This review tries to answer the question why polymers with UCST remained largely underrepresented in academic as well as applied research and what requirements have to be fulfilled to make these polymers suitable for the development of smart materials with a positive thermoresponse.
A derivation is given of the effect of a time-dependent magnetic field gradient on the spin-echo experiment, particularly in the presence of spin diffusion. There are several reasons for preferring certain kinds of time-dependent magnetic field gradients to the more usual steady gradient. If the gradient is reduced during the rf pulses, H1 need not be particularly large; if the gradient is small at the time of the echo, the echo will be broad and its amplitude easy to measure. Both of these relaxations of restrictions on the measurement of diffusion coefficients by the spin-echo technique serve to extend its range of applicability. Furthermore, a pulsed gradient can be recommended when it is critical to define the precise time period over which diffusion is being measured.
The theoretical expression derived has been verified experimentally for several choices of time dependent magnetic field gradient. An apparatus is described suitable for the production of pulsed gradients with amplitudes as large as 100 G cm−1. The diffusion coefficient of dry glycerol at 26°±1°C has been found to be (2.5±0.2)×10−8 cm2 sec−1, a value smaller than can ordinarily be measured by the steady gradient method.
Fast identification of binding activity directly from mixtures of potential ligands is possible with the NMR method described, which is based on saturation transfer to molecules in direct contact to a protein. In addition, the ligand's binding epitope is easily identified. High sensitivity and ease of use are the principal advantages of this method. The picture shows the normal 1D NMR spectrum of a mixture and the spectrum obtained by applying the STD method, which exclusively shows signals from molecules with binding affinity.
A series of copolymers of N-isopropylacrylamide (NIPA) and acryloyl amino acids were synthesized and conjugated with p-aminobenzamidine (PABA). The values of lower critical solution temperatures (LCSTs) of these polymers estimated by 1H NMR were in agreement with those estimated by the turbidometric methods. The apparent shifts in the peak positions for the polymer with increasing temperature reported in the past are shown to be artifacts resulting from the deuterium lock experiment. The 1H–1H NOESY experiments were carried out to distinguish the interactions between the protons in: (a) 6-amino caproic acid (6ACA) and NIPA and (b) PABA and NIPA. These explain the variation in the LCST of the polymers based on the contributions of the two monomers to the magnitude of the polymer–polymer interactions. The findings are further supported by proton spin–lattice relaxation time measurements.
The field of polyelectrolye–protein complexes is now benefiting from the convergence of three themes whose disciplinary distinctions formerly produced fragmentation. Theory and simulations, previously restricted to isotropic models of colloids and polyelectrolytes, are now taking into account the heterogeneity of charges in both macroions and providing far more realistic depictions of proteins. Applications of protein–polyelectrolyte systems have been strongly influenced by the opportunities presented by multilayer assembly, and the dichotomy between semi-synthetic systems and the protein–biopolyelectrolyte cognate pairs of biochemical interest has been giving way to less parochial recognition of universal effects. The junction of these disciplines, along with the development of many new methods of investigation, form the subject of this review focused on recent developments and their foundations.
The method of saturation transfer difference (STD) nuclear magnetic resonance (NMR) is an indispensable NMR tool in drug discovery. It identifies binding epitope(s) at the atomic resolution of small molecule ligands (e.g. organic drugs, peptides and oligosaccharides), while interacting with their receptors, such as proteins and/or nucleic acids. The method is widely used to screen active drug molecules, simultaneously ranking them in a qualitative way. STD NMR is highly successful for a variety of high molecular weight systems, such as whole viruses, platelets, intact cells, lipopolysaccharide micelles, membrane proteins, recombinant proteins and dispersion pigments. Modifications of STD pulse programs using (13)C and (15)N nuclei are now used to overcome the signal overlapping that occurs with more complex structures.
Because of the particular characteristics of the tumor microenvironment and tumor angiogenesis, it is possible to design drug delivery systems that specifically target anti-cancer drugs to tumors. Most of the conventional chemotherapeutic agents have poor pharmacokinetics profiles and are distributed non-specifically in the body leading to systemic toxicity associated with serious side effects. Therefore, the development of drug delivery systems able to target the tumor site is becoming a real challenge that is currently addressed. Nanomedicine can reach tumor passively through the leaky vasculature surrounding the tumors by the Enhanced Permeability and Retention effect whereas ligands grafted at the surface of nanocarriers allow active targeting by binding to the receptors overexpressed by cancer cells or angiogenic endothelial cells. This review is divided into two parts: the first one describes the tumor microenvironment and the second one focuses on the exploitation and the understanding of these characteristics to design new drug delivery systems targeting the tumor. Delivery of conventional chemotherapeutic anti-cancer drugs is mainly discussed.
Coacervation of proteins and anionic polysaccharides is both of practical and theoretical interest. From a large body of literature, it seems that the phase separation is mainly entropically driven, and may most probably be attributed to the delocalisation of the counter ions of the protein and the polysaccharide. The protein and polysaccharide appear to form complexes in solution, which can be viewed as new colloidal entities. These complex particles are neutral and exhibit an attractive interaction, which leads to a phase separation of the gas-liquid type in which a (very) dilute colloidal phase coexists with a very concentrated colloidal phase. In the case of strong poly-acids, usually, a precipitate is formed rather than a liquid coacervate phase. The structure of the concentrated polymer phase seems to resemble a continuous polymer phase in which the protein can diffuse around, as well as the individual polysaccharide molecules. Time scales of diffusion vary from milliseconds to days depending on the strength of the interaction. From a rheological point of view, the concentrated phase is much more viscous than elastic and the rheology resembles the behaviour of a (viscous) concentrated particle dispersion. Theoretical developments are limited probably due to the difficulty to describe the (correlated) charge distribution in the system. There is a strong interest in coacervates for the use of encapsulation. For the same reason, much attention is given to replacing the traditional gelatin by milk and plant proteins.
Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) is a versatile
method for studying molecular transport and exchange in solution. Stejskal-Tanner diffusion plots can be used to distinguish between chemical species even in the absence of chemical shift or T1 resolution, provided that certain conditions are satisfied. This can be used advantageously in colloidal drug delivery systems containing two drug populations, free and bound, which often differ only by their diffusion coefficients. We discuss the conditions required for the resolution of the two pools in Stejskal-Tanner plots, as well as the effects of chemical exchange. We also present two practical examples of this analysis: surfactant micelles containing the general anesthetic propofol and lipid/water liquid crystalline mesophase vehicles (cubosomes).
The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution 1H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOE's between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.
Polymers that display a physicochemical response to stimuli are widely explored as potential drug-delivery systems. Stimuli studied to date include chemical substances and changes in temperature, pH and electric field. Homopolymers or copolymers of N-isopropylacrylamide and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (known as poloxamers) are typical examples of thermosensitive polymers, but their use in drug delivery is problematic because they are toxic and non-biodegradable. Biodegradable polymers used for drug delivery to date have mostly been in the form of injectable microspheres or implant systems, which require complicated fabrication processes using organic solvents. Such systems have the disadvantage that the use of organic solvents can cause denaturation when protein drugs are to be encapsulated. Furthermore, the solid form requires surgical insertion, which often results in tissue irritation and damage. Here we report the synthesis of a thermosensitive, biodegradable hydrogel consisting of blocks of poly(ethylene oxide) and poly(L-lactic acid). Aqueous solutions of these copolymers exhibit temperature-dependent reversible gel-sol transitions. The hydrogel can be loaded with bioactive molecules in an aqueous phase at an elevated temperature (around 45 degrees C), where they form a sol. In this form, the polymer is injectable. On subcutaneous injection and subsequent rapid cooling to body temperature, the loaded copolymer forms a gel that can act as a sustained-release matrix for drugs.
Environmentally sensitive hydrogels have enormous potential in various applications. Some environmental variables, such as low pH and elevated temperatures, are found in the body. For this reason, either pH-sensitive and/or temperature-sensitive hydrogels can be used for site-specific controlled drug delivery. Hydrogels that are responsive to specific molecules, such as glucose or antigens, can be used as biosensors as well as drug delivery systems. Light-sensitive, pressure-responsive and electro-sensitive hydrogels also have the potential to be used in drug delivery and bioseparation. While the concepts of these environment-sensitive hydrogels are sound, the practical applications require significant improvements in the hydrogel properties. The most significant weakness of all these external stimuli-sensitive hydrogels is that their response time is too slow. Thus, fast-acting hydrogels are necessary, and the easiest way of achieving that goal is to make thinner and smaller hydrogels. This usually makes the hydrogel systems too fragile and they do not have mechanical strength necessary in many applications. Environmentally sensitive hydrogels for drug delivery applications also require biocompatibility. Synthesis of new polymers and crosslinkers with more biocompatibility and better biodegradability would be essential for successful applications. Development of environmentally sensitive hydrogels with such properties is a formidable challenge. If the achievements of the past can be extrapolated into the future, however, it is highly likely that responsive hydrogels with a wide array of desirable properties can be made.
Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.
We report here the synthesis and self-assembly of a series of three molecules with dendron rodcoil architecture that contain conjugated segments of oligo(thiophene), oligo(phenylene-vinylene), and oligo(phenylene). Despite their structural differences, all three molecules yield similar self-assembled structures. Electron and atomic force microscopy reveals the self-assembly of the molecules into high aspect ratio ribbon-like nanostructures which at low concentrations induce gelation in nonpolar solvent. Self-assembly results in a blue-shifted absorption spectrum and a red-shifted, quenched fluorescence spectrum, indicating aggregation of the conjugated segments within the ribbon-like structures. The assembly of these molecules into one-dimensional nanostructures is a route to pi-pi stacked supramolecular polymers for organic electronic functions. In the oligo(thiophene) derivative, self-assembly leads to a 3 orders of magnitude increase in the conductivity of iodine-doped films due to self-assembly. We also found that electric field alignment of these supramolecular assemblies can be used to create arrays of self-assembled nanowires on a device substrate.
We now report the formation mechanism of the thermoresponsive-type coacervate with the novel functional temperature-sensitive polymer, poly(N-isopropylacrylamide-co-2-hydroxyisopropylacrylamide) (poly(NIPAAm-co-HIPAAm)), synthesized in our laboratory. The effects of introducing the hydrophilic comonomer (HIPAAm) into the copolymer chains and adding salts on the behaviors of the coacervate droplets induced in the poly(NIPAAm-co-HIPAAm) aqueous solutions were investigated. Not only the particle sizes of the coacervate droplets but also the cloud points of the copolymer solutions could be modulated by the HIPAAm content incorporated in the copolymers. Moreover, the particle sizes of the coacervate droplets were also changed by adding salts. Namely, the particle sizes increased with the decreasing HIPAAm composition and increasing NaCl concentration. In addition, the 1H NMR and differential scanning calorimetric measurements suggested that as the HIPAAm content decreased or NaCl concentration increased, dehydration of the copolymers induced in the phase transition and/or separation became much easier. Therefore, on the basis of the findings obtained from these measurements, we determined that the particle sizes of the coacervate droplets induced in the temperature-sensitive polymers increased as the number of the water molecules, which are dissociated from the polymeric chains during the phase transition and/or separation, increased. Besides, to examine the separation of the model solutes, the aqueous two-phase separation with the coacervate droplets of poly(NIPAAm-co-HIPAAm) was carried out. The partitions of Methyl Orange as a model solute under both acidic (pH 2) and basic (pH 12) conditions were performed. The amount of Methyl Orange partitioned into the coacervate droplets at pH 12 is much greater than that at pH 2, which indicated that the coacervate droplets could recognize a slight difference in the polarity or structure between the model solutes.
Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics, and interactions of smaller macromolecules (< approximately 25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems.
Biodegradable Block Copolymers as Injectable Drug-Delivery Systems
- B Jeong
- Y H Bae
- D S Lee
- S W Kim
Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Biodegradable Block
Copolymers as Injectable Drug-Delivery Systems. Nature 1997, 388
(6645), 860−862.