Polyelectrolyte Nanocomplexes Research Articles

A Critical Review on Glycosaminoglycan Derived Polymers as a Novel Drug Delivery System in Tissue Engineering: Recent Advancement and Clinical Application

Glycosaminoglycans (GAGs), natural components of the extracellular matrix, exert significant influence over cellular function and regulate the microenvironment surrounding cells. This characteristic makes them promising targets for therapeutic intervention across a spectrum of diseases. In the realm of medical research, there has been a longstanding quest for precise and targeted drug delivery methods to mitigate adverse effects and enhance the efficacy of treatments for conditions, such as wounds, cancer, and organ disorders. However, implementing a systemic delivery approach, particularly for protein-based therapeutics, poses challenges. Addressing this challenge requires the development of biocompatible materials capable of efficiently encapsulating and releasing therapeutic proteins. GAGs emerge as promising candidates possessing these desirable attributes, given their bioderived nature and ability to modulate biological responses. Within the realm of GAGs, various linear polysaccharides exhibit diverse functionalities and payloads. Notably, hyaluronic acid (HA) and chondroitin sulfate (CS) have been utilized as polysaccharide-based biomaterials for drug delivery, particularly in the treatment of rheumatoid arthritis. Modified HA and CS can self-assemble into micelles or micellar nanoparticles (NPs), enabling precise and controlled drug delivery. This paper explores a range of NP formulations derived from HA and CS, including drug conjugates, polymers, small molecules, polyelectrolyte nanocomplexes (PECs), metals, and nanogels. The versatility of these NP formulations extends to various therapeutic applications, including cancer chemotherapy, gene therapy, photothermal therapy (PTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and immunotherapy. By harnessing the unique properties of HA and CS, these NP-based systems offer promising avenues for advancing therapeutic interventions in diverse clinical settings.

Coordinated modulation of long non-coding RNA ASBEL and curcumin co-delivery through multicomponent nanocomplexes for synchronous triple-negative breast cancer theranostics

BackgroundAbnormally regulated long non-coding RNAs (lncRNAs) functions in cancer emphasize their potential to serve as potential targets for cancer therapeutic intervention. LncRNA ASBEL has been identified as oncogene and an anti-sense transcript of tumor-suppressor gene of BTG3 in triple-negative breast cancer (TNBC).ResultsHerein, multicomponent self-assembled polyelectrolyte nanocomplexes (CANPs) based on the polyelectrolytes of bioactive hyaluronic acid (HA) and chitosan hydrochloride (CS) were designed and prepared for the collaborative modulation of oncogenic lncRNA ASBEL with antago3, an oligonucleotide antagonist targeting lncRNA ASBEL and hydrophobic curcumin (Cur) co-delivery for synergetic TNBC therapy. Antago3 and Cur co-incorporated CANPs were achieved via a one-step assembling strategy with the cooperation of noncovalent electrostatic interactions, hydrogen-bonding, and hydrophobic interactions. Moreover, the multicomponent assembled CANPs were ulteriorly decorated with a near-infrared fluorescence (NIRF) Cy-5.5 dye (FCANPs) for synchronous NIRF imaging and therapy monitoring performance. Resultantly, MDA-MB-231 cells proliferation, migration, and invasion were efficiently inhibited, and the highest apoptosis ratio was induced by FCANPs with coordination patterns. At the molecular level, effective regulation of lncRNA ASBEL/BTG3 and synchronous regulation of Bcl-2 and c-Met pathways could be observed.ConclusionAs expected, systemic administration of FCANPs resulted in targeted and preferential accumulation of near-infrared fluorescence signal and Cur in the tumor tissue. More attractively, systemic FCANPs-mediated collaborative modulating lncRNA ASBEL/BTG3 and Cur co-delivery significantly suppressed the MDA-MB-231 xenograft tumor growth, inhibited metastasis and extended survival rate with negligible systemic toxicity. Our present study represented an effective approach to developing a promising theranostic platform for combating TNBC in a combined therapy pattern.

Codelivery of synergistic antimicrobials with polyelectrolyte nanocomplexes to treat bacterial biofilms and lung infections.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

Nanocomplex of quaternized cyclodextrin grafted chitosan and hyaluronic acid for a skin delivery

Water soluble quaternized cyclodexrin grafted chitosan (QCD-g-CS) was synthesized by combining both beneficial properties of β-cyclodextrin (β-CD) and the chitosan (CS) backbone. The chitosan backbone exhibits positive charges, while the β-CD moieties are available to include hydrophobic guest molecules into the cavity. The present work demonstrates a formation of nanocomplexes by simple mixing of the cationic QCD-g-CS with three different molecular weights of anionic Hyaluronic acid (low, medium and high HA; LHA, MHA and HHA, respectively). The HA is well-known on providing hydration to the skin and normalize keratinization. However, its strong hydrophilicity limits skin absorption. The polyelectrolyte nanocomplexes between QCD-g-CS and HA formed through the electrostatic interactions were confirmed by FTIR. Particle size of HA nanocomplexes were greater than that of free QCD-g-CS and increased with an increase in HA content. The complex of LHA and MHA improve the water retention capacity as well as ability to control the release of HA to be slower than the original HA. The release of both LHA and MHA from their complexes were both limited diffusion kinetics. Pronounced effect of small particle sizes of LHA complexes was found to benefit skin penetration. Clinical study indicated that LHA complexes improved skin texture and elasticity due to an increase in skin hydration. It is suggested that the QCD-g-CS in combination with anionic hydrophilic HA can be used as a promising polysaccharide-based skin delivery system.

Hybrid Polyelectrolyte Nanocomplexes for Non-Viral Gene Delivery with Favorable Efficacy and Safety Profile.

The development of nanovectors for precise gene therapy is increasingly focusing on avoiding uncontrolled inflammation while still being able to effectively act on the target sites. Herein, we explore the use of non-viral hybrid polyelectrolyte nanocomplexes (hPECs) for gene delivery, which display good transfection efficacy coupled with non-inflammatory properties. Monodisperse hPECs were produced through a layer-by-layer self-assembling of biocompatible and biodegradable polymers. The resulting nanocomplexes had an inner core characterized by an EGFP-encoding plasmid DNA (pDNA) complexed with linear polyethyleneimine or protamine (PEI or PRM) stabilized with lecithin and poly(vinyl alcohol) (PVA) and an outer layer consisting of medium-molecular-weight chitosan (CH) combined with tripolyphosphate (TPP). PEI- and PRM-hPECs were able to efficiently protect the genetic cargo from nucleases and to perform a stimuli-responsive release of pDNA overtime, thus guaranteeing optimal transfection efficiency. Importantly, hPECs revealed a highly cytocompatible and a non-inflammatory profile in vitro. These results were further supported by evidence of the weak and unspecific interactions of serum proteins with both hPECs, thus confirming the antifouling properties of their outer shell. Therefore, these hPECs represent promising candidates for the development of effective, safe nanotools for gene delivery.

Identification of inflammatory regulation roles of thalidomide/ruxolitinib in nucleus pulposus and construction of polyelectrolyte nanocomplexes-impregnated injectable hydrogels for synergistic intervertebral disk degeneration treatment

Degeneration of the intervertebral discs (IVD) is an important underlying etiology of degenerative diseases contributor to lower back and neck pain. Alleviation of inflammation is recognized as the most direct way for the treatment of IVD. Repurposing of approved drugs is a viable alternative strategy to de novo drug discovery and development. The aim of this study is to identify the inflammatory regulation roles of the small molecule thalidomide (an immunomodulatory and TNF-α inhibitor) and ruxolitinib (a selective JAK1/2 inhibitor) in nucleus pulposus (NP) cells and investigate the combinational therapeutic potentials of thalidomide and ruxolitinib for ameliorating IVD degeneration. RNA-sequencing (RNA-seq) was used to detect differentially expressed genes (DEGs) after treatment of human NP cells with individual thalidomide and ruxolitinib, and relevant signaling pathways and functions of DEGs were detected by enrichment analysis. Western blot and immunofluorescence were applied to explore the mechanisms by which thalidomide and ruxolitinib regulated inflammatory factors in human NP cells. Moreover, we developed ruxolitinib and thalidomide co-delivered polyelectrolyte nanocomplexes (RTNPs) by the assembly of chitosan derivatives of positively charged chitosan hydrochloride (CS) and negatively charged carboxymethyl chitosan (CMCS). In vitro studies revealed that RTNPs could efficiently deliver drugs into NP cells and corresponding extracellular matrix (ECM)-related genes and proteins were adjusted. Furthermore, The RTNPs were assembled with Pluronic F127 to form a RTNPs/F127 injectable thermoresponsive hydrogels to maintain resident time within the nucleus pulposus, achieve sustained drugs release and achieve long-term therapy effects. The caudal disc degeneration model of SD rats was established by acupuncture, and X-ray, magnetic resonance scanning, μCT scanning, and histological methods were performed to detect the therapeutic effects of RTNPs/F127 hydrogel on IVD in vivo. As was demonstrated in the in vivo studies, in situ intradiscal injection of RTNPs/F127 composite displayed a relatively slower disc degenerating progression in a puncture-induced IVD rat model compared with the monotherapy pattern.

Drug resistance reversal by interventing cancer bioenergetics with spherical helical polypeptide-potented gene silencing

Drug resistance diminishes the efficacies of anticancer therapeutics and causes the failure of clinical cancer chemotherapy. Herein, we reported a promising strategy to reverse drug resistance via RNA interference (RNAi)-assisted intervention of cancer bioenergetics. Densely packed, guanidine-rich, spherical helical polypeptide (DPP) was developed to encapsulate doxorubicin (DOX) inside its hydrophobic cavity and form polyelectrolyte nanocomplexes (NCs) with PKM2 siRNA. The NCs were further surface-decorated with hyaluronic acid (HA) to enhance the serum stability and promote tumor targeting via binding to over-expressed CD44 on cancer cell membranes. DPP with multivalent topology mediated potent trans-membrane delivery of PKM2 siRNA into DOX-resistance cancer cells. PKM2 down-regulation suppressed the glycolysis metabolism and depleted energy supply for ATP-binding cassette (ABC) transporters, thus inhibiting drug efflux to reverse drug resistance. Meanwhile, PKM2 silencing starved tumor cells, induced intracellular reactive oxygen species (ROS) generation, and triggered mitochondrial collapse, which aggravated cell apoptosis to achieve potent and cooperative anti-cancer effect with chemotherapy. This study thus provides an effective topology-controlled approach for designing trans-membrane siRNA delivery vehicles, and it also renders a promising tool in rejuvenating chemotherapy against drug-resistant tumors via a unique anti-bioenergetics mechanism.

Polyelectrolyte nanocomplexes based on chitosan derivatives for wound healing application

Wound healing, when compromised, may be guided by biological cues such as Arg-Gly-Asp (RGD), a peptide known to induce cell adhesion and migration, eventually combined with adapted nanocarriers. Three different formulations were prepared and investigated in vitro for topical application. All formulations were based on carboxylated and trimethylated chitosan (CMTMC) displaying RGD. The polyelectrolyte nanocomplexes were prepared by mixing two oppositely charged polymers of CMTMC and chondroitin sulfate at different polymer ratios and subsequently characterized by dynamic light scattering and scanning electron microscopy. Hydrogels and foams with a high concentration of RGD-functionalized chitosan (3%) and hyaluronic acid (1.5%) that formed gel-embedded nanocomplexes were developed. In vitro assays showed absence of toxicity, ability to promote proliferation over 7 days and promotion of migration of human dermal fibroblasts treated with any of our formulations. These formulations were shown to be suitable for easy topical application and have the potential to accelerate wound healing.

Hydrolytically Degradable PEGylated Polyelectrolyte Nanocomplexes for Protein Delivery.

Novel oppositely charged polyphosphazene polyelectrolytes containing grafted poly(ethylene glycol) (PEG) chains were synthesized as modular components for the assembly of biodegradable PEGylated protein delivery vehicles. These macromolecular counterparts, which contained either carboxylic acid or tertiary amino groups, were then formulated at near physiological conditions into supramolecular assemblies of nanoscale level, below 100 nm. Nanocomplexes with electroneutral surface charge, as assessed by zeta potential measurements, were stable in aqueous solutions, which suggests their compact polyelectrolyte complex "core"-hydrophilic PEG "shell" structure. Investigation of PEGylated polyphosphazene nanocomplexes as agents for noncovalent PEGylation of the therapeutic protein l-asparaginase (L-ASP) in vitro demonstrated their ability to dramatically reduce protein antigenicity, as measured by antibody binding using enzyme linked immunosorbent assay (ELISA). Encapsulation in nanocomplexes did not affect enzymatic activity of L-ASP, but improved its thermal stability and proteolytic resistance. Gel permeation chromatography (GPC) experiments revealed that all synthesized polyphosphazenes exhibited composition controlled hydrolytic degradability in aqueous solutions at neutral pH and showed greater stability at lower temperatures. Overall, novel hydrolytically degradable polyphosphazene polyelectrolytes capable of spontaneous self-assembly into PEGylated nanoparticulates in aqueous solutions can potentially enable a simple and effective approach to modifying therapeutic proteins without the need for their covalent modification.

Composite carbohydrate interpenetrating polyelectrolyte nano-complexes (IPNC) as a controlled oral delivery system of citalopram HCl for pediatric use: in-vitro/in-vivo evaluation and histopathological examination.

Citalopram HCl (CH) is one of the few drugs which can be used safely in childhood psychiatric disorders. This study was focused on the preparation of interpenetrating polyelectrolytes nano-complexes (IPNC) to transform the hydrophilic carbohydrate polymers into an insoluble form. The IPNCs were loaded with CH to sustain its effect. The IPNC2 (composed of chitosan:pectin in a 3:1 ratio) showed the most extended drug release pattern (P< 0.05) and followed a Higuchi-order kinetics model. It was characterized using SEM, X-rays diffractometry, and FTIR. In-vivo studies were performed using immature rats with induced depression, and were based on the investigation of behavioral, biochemical, and histopathological changes at different time intervals up to 24h. Rats treated with IPNC2 showed a significant more rapid onset of action and more extended effect in the behavioral tests, in addition to a significantly higher serotonin brain level up to 24h, compared to rats treated with the market product (P< 0.05). The histopathological examination showed a profound amelioration of the cerebral cortex features of the depressed rats after IPNC2 administration. This study proves the higher efficacy and more extended effect of the new polyelectrolytes nano-complexes compared to the market product.

Characterization of high density lipoprotein from egg yolk and its ability to form nanocomplexes with chitosan as natural delivery vehicles

In this work, egg yolk high density lipoprotein (HDL) was first extracted and solubilized with a salt-free method. The freshly purified egg yolk HDL was characterized for its physicochemical properties and its delivery potentials for hydrophobic bioactives were also investigated. Native chitosan (NACS) and its amphiphilic derivative (stearic acid conjugated chitosan, SACS) were adopted to fabricate polyelectrolyte nanocomplexes with egg yolk HDL and the fabrication as well as formulation of nanocomplexes were optimized. HDL/SACS and HDL/NACS nanocomplexes had a diameter of 75 nm and 97 nm, respectively. Gastrointestinal stability tests suggested that the nanocomplexes were more stable in gastric phase than in the intestinal condition with the presence of digestive enzymes (pepsin and pancreatin). Curcumin was selected as a model hydrophobic compound to assess the encapsulation and delivery potentials of HDL-based nanocomplexes. HDL/SACS nanocomplexes showed better encapsulation property than HDL/NACS nanocomplexes due to enhanced hydrophobic interactions, which was confirmed by fluorescence and surface hydrophobicity tests. The HDL/SACS nanocomplexes also exhibited slower sustained release of curcumin in simulgated GI tract, indicating the promising features as nanoscale oral delivery vehicles for hydrophobic phytochemicals.

Optimisation of the self-assembly process: production of stable, alginate-based polyelectrolyte nanocomplexes with protamine

The aim of this work was to investigate the possibility of covalent cross-linker-free, polyelectrolyte complex formation at the nanoscale between alginic acid (as sodium alginate, ALG) and protamine (PROT). Optimisation of the self-assembly conditions was performed by varying the type of polymer used, pH of component solutions, mass mixing ratio of the components and the speed and order of component addition on the properties of complexes. Homogenous particles with nanometric sizes resulted when an aqueous dispersion of ALG was rapidly mixed with a solution of PROT. The polyelectrolyte complex between ALG and PROT was confirmed by infrared spectroscopy. To facilitate incorporation of drugs soluble at low pH, pH of ALG dispersion was decreased to 2; however, no nanoparticles (NPs) were formed upon complexation with PROT. Adjusting pH of PROT solution to 3 resulted in the formation of cationic or anionic NPs with a size range 70–300 nm. Colloidal stability of selected alginic acid low/PROT formulations was determined upon storage at room temperature and in liquid media at various pH. Physical stability of NPs correlated with the initial surface charge of particles and was time- and pH-dependent. Generally, better stability was observed for anionic NPs stored as native dispersions and in liquids covering a range of pH.

Triggered Release of Encapsulated Cargo from Photoresponsive Polyelectrolyte Nanocomplexes.

Combining the numerous advantages of using light as a stimulus, simple free radical random copolymerization, and the easy, all-aqueous preparation of polyelectrolyte complexes (PECs), we prepared photolabile PEC nanoparticles and demonstrated their rapid degradation under UV light. As a proof of concept demonstration, the dye Nile Red was encapsulated in the PECs and successfully released into the surrounding solution as the polyelectrolyte nanocomplex carriers dissolved upon light irradiation.

Zinc-stabilized colloidal polyelectrolyte complexes of chitosan/hyaluronan: a tool for the inhibition of HIV-1 infection.

Zinc(ii) stabilized polyelectrolyte nano-complexes (PECs) of chitosan and hyaluronan (HYA) were designed as safe and efficient drug delivery systems. HIV-1 reverse transcriptase inhibitor tenofovir (TF) was quantitatively encapsulated and the particle interface could be functionalized in PBS with targeting proteins such as anti-α4β7 immunoglobulin A. Chitosan-HYA nanoPECs were non-cytotoxic on human peripheral blood mononuclear cells (PBMCs), within the investigated nanoparticle concentrations. A dose-dependent reduction of the HIV-1 infection of PBMCs co-cultured with the nanocarriers was observed. Even more interestingly, a synergistic effect was evidenced with the nanocarriers by comparing the IC50 (50% inhibitory concentration) value of the aqueous TF solution (4.35 μmol L-1) with that of TF loaded nanoPECs (1.71 μmol L-1) and anti-α4β7 IgA functionalized TF/nanoPECs (1.01 μmol L-1). This effect could be attributed to the presence of zinc(ii) in the formulation of the colloids. All these data establish that the zinc(ii) stabilized chitosan-HYA nanoPECs can be potentially efficient and safe colloidal delivery system candidates for enhancing antiviral activities in the treatment of HIV infection and AIDS.

Development and preclinical pharmacokinetics of a novel subcutaneous thermoresponsive system for prolonged delivery of heparin

Heparin is still widely used for treatment and prevention of thromboembolic diseases. Due to specific physicochemical properties, it requires frequent parenteral injections. In this study we present the development and in vitro evaluation of an advanced delivery system for prolonged subcutaneous release of heparin. The delivery system consisted of an in situ forming thermoresponsive poloxamer-based platform combined with pH-responsive polyelectrolyte heparin/chitosan nanocomplexes. Thermoresponsive hydrogels were tested for gelation temperature, gel dissolution and in vitro heparin release, whereas polyelectrolyte nanocomplexes were physico-chemically characterized, as well as tested for in vitro cytotoxicity and in vitro heparin release. Hydrogel combined of two poloxamers demonstrated the highest gelation temperature (28.6°C), while the addition of hydroxypropyl methylcellulose prolonged gel dissolution. On the other hand, nanocomplexes’ dispersions, prepared at 1:1 heparin/chitosan mass ratio and in the concentration range 0.375–1.875mg/mL, demonstrated mean diameter <400nm and zeta potential >34mV. Pharmacokinetics of selected formulations (thermoresponsive hydrogel, nanocomplexes and a dual system consisting of nanocomplexes incorporated into thermoresponsive hydrogel) were studied in rats. Heparin plasma concentration-time profiles revealed a double-peak phenomenon, probably due to heparin diffusion inside the polymer matrix and gel dissolution. Pharmacokinetic parameters were determined by a non-linear mixed effects modeling approach. It was demonstrated that thermoresponsive hydrogel with heparin/chitosan nanocomplexes enabled the lowest absorption rate of heparin into systemic circulation and provided heparin concentration above the prophylaxis threshold for 5 days. In situ gelling thermoresponsive matrix combined with chitosan nanocomplexes present a promising delivery system for heparin, requiring less frequent administration during long-term treatment.

Covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination

The physical stability of polyelectrolyte nanocomplexes composed of trimethyl chitosan (TMC) and hyaluronic acid (HA) is limited in physiological conditions. This may minimize the favorable adjuvant effects associated with particulate systems for nasal and intradermal immunization. Therefore, covalently stabilized nanoparticles loaded with ovalbumin (OVA) were prepared with thiolated TMC and thiolated HA via ionic gelation followed by spontaneous disulfide formation after incubation at pH 7.4 and 37 °C. Also, maleimide PEG was coupled to the remaining thiol-moieties on the particles to shield their surface charge. OVA-loaded TMC/HA nanoparticles had a size of around 250–350 nm, a positive zeta potential and OVA loading efficiencies up to 60%. Reacting the thiolated particles with maleimide PEG resulted in a slight reduction of zeta potential (from + 7 to + 4 mV) and a minor increase in particle size. Stabilized TMC-S-S-HA particles (PEGylated or not) showed superior stability in saline solutions compared to non-stabilized particles (composed of nonthiolated polymers) but readily disintegrated upon incubation in a saline buffer containing 10 mM dithiothreitol. In both the nasal and intradermal immunization study, OVA loaded stabilized TMC-S-S-HA particles demonstrated superior immunogenicity compared to non-stabilized particles (indicated by higher IgG titers). Intranasal, PEGylation completely abolished the beneficial effects of stabilization and it induced no enhanced immune responses against OVA after intradermal administration. In conclusion, stabilization of the TMC/HA particulate system greatly enhances the immunogenicity of OVA in nasal and intradermal vaccination.

Thiolated trimethyl chitosan nanocomplexes as gene carriers with high in vitro and in vivo transfection efficiency

Trimethyl chitosan–cysteine conjugate (TMC–Cys) was evaluated as non-viral gene carriers to combine the advantages of TMC and thiolated chitosan. TMC–Cys with various molecular weights (30, 100, and 200 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanocomplexes with plasmid encoding enhanced green fluorescence protein (pEGFP), which demonstrated preferable diameters of below 200 nm and zeta potentials of + 15 to + 20 mV. Cell binding and uptake of TMC–Cys/pEGFP nanocomplexes (TMC–Cys NC) were enhanced 2.4–3.0 and 1.4–3.0 folds, respectively, compared to TMC/pEGFP nanocomplexes (TMC NC). pEGFP could be easily released from TMC–Cys NC at the intracellular glutathione concentration, which promoted its nuclear transport and accumulation. Consequently, TMC–Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC–Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC–Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively. Therefore, TMC–Cys/DNA nanocomplexes could be a promising gene delivery system with in vitro and in vivo superiority to Lipofectamine2000.

Chitosan–plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Self-assembled polyelectrolyte nanocomplexes between chitosan derivatives and enoxaparin

Polyelectrolyte complexes (PEC) formed from chitosan derivatives and enoxaparin were prepared and parameters influencing complex formation were characterized. Dynamic light scattering (DLS) and laser doppler anemometry (LDA) were used to study the complexation process. Surface morphology of the PECs was observed with atomic force microscopy (AFM). The PEC formation process was influenced by a variety of parameters, including the system pH, polymer/enoxaparin mass ratio, polymer molecular weight, concentration and structure. Soluble complexes in the size range of 200–500 nm with spherical morphology could be obtained at optimized polymer/enoxaparin ratios in the pH range of 3.0–6.5, with positive charge and drug encapsulation efficiency of approximately 90%. An increase in ionic strength of the medium accelerated the dissociation of chitosan/enoxaparin complexes. In contrast, chitosan thiolation, methylation and PEGylation significantly improved the stability of the complexes. Physicochemical properties of the PECs, including particle size, charge density and morphology, could be modified by using different chitosan derivatives. On the basis of our results, we suggest that interactions involved in PEC formation were partly electrostatic in nature, involving the positively charged chitosan derivatives and the negatively charged enoxaparin at pH values in the vicinity of the p K a interval of the two polymers. Oral absorption of the polyelectrolyte nanocomplexes will be studied in vivo.

Peroral delivery of insulin using chitosan derivatives: A comparative study of polyelectrolyte nanocomplexes and nanoparticles

Polymeric delivery systems based on nanoparticles (NP) have emerged as a promising approach for peroral insulin delivery. Using a trimethyl chitosan (TMC) and a PEG- graft-TMC copolymer, polyelectrolyte complexes (PEC) and nanoparticles were prepared and their properties were compared. The amount of insulin was quantified by HPLC and the stability of PEC and NP upon exposure to simulated gastrointestinal (GI) fluid was monitored by dynamic laser light scattering. It was shown that polymer/insulin (+/−) charge ratio played an important role in PEC and NP formation. Stable, uniform, and spherical PEC/NP with high insulin association efficiency (AE) were formed at or close to optimized polymer/insulin (+/−) charge ratio, depending on polymer structure. All PEC were more stable in pH 6.8 simulated intestinal fluid (SIF) than NP. The PEC also appeared to play some role in protecting insulin from degradation at higher temperature and with proteolytic enzyme more efficiently than NP. On the basis of these results, polyelectrolyte complexation can be suggested as a potentially useful technique for generating insulin delivery systems for peroral administration.