siRNA nanocarriers based on methacrylic acid copolymers

Poly(ethylene glycol)-b-poly(propyl methacrylate-co-methacrylic acid) (PEG-b-P(PrMA-co-MAA) can be complexed with poly(amido amine) (PAMAM) dendrimers and nucleic acids to form pH-responsive nanosized core-shell type polyion complex micelles (PICMs). These PICMs have the ability to lose their shell and release the PAMAM/nucleic acid core under mildly acidic conditions such as those encountered in the endosomal compartment. In this work, pH-sensitive PICMs composed of PEG-b-P(PrMA-co-MAA), different PAMAMs, and siRNAs were prepared and characterized. These micelles had mean diameters ranging from 50 to 100 nm depending on the structure of the polycationic component. In order to trigger PICM uptake by receptor-mediated endocytosis, the micelles were decorated with an antibody fragment directed against the transferrin receptor (anti-CD71). The targeting ligand was stably conjugated to a semi-telechelic amino-PEG-b-P(PrMA-co-MAA) via a maleimide/activated ester bifunctional linker, yielding up to 60%-80% functionalization of the maleimide groups. The cellular uptake of the micelles was assessed on human prostate cancer cells (PC-3) via flow cytometry. Native PICMs and micelles bearing a non-specific antibody fragment were taken up to the same extent with a low efficiency, whereas anti-CD71 Fab′-decorated PICMs exhibited significantly higher uptake. The capacity of the targeted, siRNA-loaded, PICMs to downregulate the expression of the Bcl-2 anti-apoptotic oncoprotein was investigated using the appropriate unmodified or 2′-modified (2′F-RNA and 2′F-ANA) siRNA sequence. Bcl-2 mRNA and protein levels were greatly reduced when the cells were transfected with anti-CD71 decorated PICMs. Optimal silencing was achieved with the chemically modified siRNA. These data suggest that combining optimized siRNA chemistry with an effective delivery system can potentiate the activity of siRNA, thereby potentially reducing the total dose of carrier required to achieve a pharmacological effect.     

PEG conjugated VEGF siRNA for anti-angiogenic gene therapy.

A novel siRNA delivery system based on polyelectrolyte complex (PEC) micelles was introduced in this study. Vascular endothelial growth factor (VEGF) siRNA was conjugated to poly(ethylene glycol) (PEG) via a disulfide linkage (siRNA-PEG). The siRNA-PEG conjugate could form PEC micelles by interacting with cationic polyethylenimine (PEI) as a core forming agent. The VEGF siRNA-PEG/PEI PEC micelles showed greater stability than naked VEGF siRNA against enzymatic degradation. Under a reductive condition similar to cytosolic environment, an intact form of siRNA was released from the siRNA-PEG conjugate by cleavage of the disulfide linkage. The VEGF siRNA-PEG/PEI PEC micelles effectively silenced VEGF gene expression in prostate carcinoma cells (PC-3) up to 96.5% under an optimized formulation condition. They also showed a far superior VEGF gene silencing effect than VEGF siRNA/PEI complexes even in the presence of serum. This study suggests that the siRNA delivery system using VEGF siRNA-PEG/PEI PEC micelles could be potentially applied to RNAi-based anti-angiogenic treatment of cancer in vivo.     

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0 kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2 kDa PEI (~ 2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25 kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.     

Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer

The co-delivery of drug combination at a controlled ratio via the same vehicle to the cancer cells is offering the advantages such as spatial–temporal synchronization of drug exposure, synergistic therapeutic effects and increased therapeutic potency. In an attempt to develop such multidrug vehicle this work focuses on functional biodegradable and biocompatible polypeptide-based polymeric micelles. Triblock copolymers containing the blocks of ethylene glycol, glutamic acid and phenylalanine (PEG–PGlu–PPhe) were successfully synthesized via NCA-based ring-opening copolymerization and their composition was confirmed by ¹H NMR. Self-assembly behavior of PEG–PGlu₉₀–PPhe₂₅ was utilized for the synthesis of hybrid micelles with PPhe hydrophobic core, cross-linked ionic PGlu intermediate shell layer, and PEG corona. Cross-linked (cl) micelles were about 90 nm in diameter (ξ-potential = − 20 mV), uniform (narrow size distribution), and exhibited nanogels-like behavior. Degradation of cl-micelles was observed in the presence of proteolytic enzymes (cathepsin B). The resulting cl-micelles can incorporate the combination of drugs with very different physical properties such as cisplatin (15 w/w% loading) and paclitaxel (9 w/w% loading). Binary drug combination in cl-micelles exhibited synergistic cytotoxicity against human ovarian A2780 cancer cells and exerted a superior antitumor activity by comparison to individual drug-loaded micelles or free cisplatin in cancer xenograft model in vivo. Tunable composition and stability of these hybrid biodegradable micelles provide platform for drug combination delivery in a broad range of cancers.     

Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers.

Poly(lactic-co-glycolic acid) (PLGA)-grafted poly(L-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200-300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.     

Biocompatible gelatin nanoparticles for tumor-targeted delivery of polymerized siRNA in tumor-bearing mice

Structural modifications of the siRNA backbone improved its physiochemical properties for incorporating in gene carriers without loss of gene-silencing efficacy. These modifications provide a wider variety of choice of vector systems for siRNA delivery. We developed a tumor-targeted siRNA delivery system using polymerized siRNA (poly-siRNA) and natural polymer gelatin. The polymerized siRNA (poly-siRNA) was prepared through self-polymerization of thiol groups at the 5′-end of sense and anti-sense strands of siRNA and was encapsulated in the self-assembled thiolated gelatin (tGel) nanoparticles (NPs) with chemical cross-linking. The resulting poly-siRNA-tGel (psi-tGel) nanoparticles (average of 145 nm in diameter) protect siRNA molecules from enzymatic degradation, and can be reversibly reduced to release functional siRNA molecules in reductive conditions. The psi-tGel NPs presented efficient siRNA delivery in red fluorescence protein expressing melanoma cells (RFP/B16F10) to down-regulate target gene expression. In addition, the NPs showed low toxicity at a high transfection dose of 125 μg/ml psi-tGel NPs, which included 1 μM of siRNA molecules. In tumor-bearing mice, the psi-tGel NPs showed 2.8 times higher tumor accumulation than the naked poly-siRNA, suggesting tumor-targeted siRNA delivery of psi-tGel NPs. Importantly, the psi-tGel NPs induced effective tumor RFP gene silencing in vivo without remarkable toxicity. The psi-tGel NPs have great potential for a systemic siRNA delivery system for cancer therapy, based on their characteristics of low toxicity, tumor accumulation, and effective siRNA delivery.     

Tuning core vs. shell dimensions to adjust the performance of nanoscopic containers for the loading and release of doxorubicin

Detailed studies were performed to probe the effects of the core and shell dimensions of amphiphilic, shell crosslinked, knedel-like polymer nanoparticles (SCKs) on the loading and release of doxorubicin (DOX), a widely-used chemotherapy agent, in aqueous buffer, as a function of the solution pH. Effects of the nanoparticle composition were held constant, by employing SCKs constructed from a single type of amphiphilic diblock copolymer, poly(acrylic acid)-b-polystyrene (PAA-b-PS). A series of four SCK nanoparticle samples, ranging in number-average hydrodynamic diameter from 14-30 nm, was prepared from four block copolymers having different relative block lengths and absolute degrees of polymerization. The ratios of acrylic acid to styrene block lengths ranged from 0.65 to 3.0, giving SCKs with ratios of shell to core volumes ranging from 0.44 to 2.1. Although the shell thicknesses were calculated to be similar (1.5-3.1 nm by transmission electron microscopy (TEM) calculations and 3.5-4.9 nm by small angle neutron scattering (SANS) analyses), two of the SCK nanoparticles had relatively large core diameters (19 ± 2 and 20 ± 2 nm by TEM; 17.4 and 15.3 nm by SANS), while two had similar, smaller core diameters (11 ± 2 and 13 ± 2 nm by TEM; 9.0 and 8.9 nm by SANS). The SCKs were capable of being loaded with 1500-9700 DOX molecules per each particle, with larger numbers of DOX molecules packaged within the larger core SCKs. Their shell-to-core volume ratio showed impact on the rates and extents of release of DOX, with the volume occupied by the poly(acrylic acid) shell relative to the volume occupied by the polystyrene core correlating inversely with the diffusion-based release of DOX. Given that the same amount of polymer was used to construct each SCK sample, SCKs having smaller cores and higher acrylic acid vs. styrene volume ratios were present at higher concentrations than were the larger core SCKs, and gave lower final extents of release., Higher final extents of release and faster rates of release were observed for all DOX-loaded particle samples at pH 5.0 vs. pH 7.4, respectively, ca. 60% vs. 40% at 60 h, suggesting promise for enhanced delivery within tumors and cells. By fitting the data to the Higuchi model, quantitative determination of the kinetics of release was made, giving rate constants ranging from 0.0431 to 0.0540 h^− 1/2 at pH 7.4 and 0.106 to 0.136 h^− 1/2 at pH 5.0. In comparison, the non-crosslinked polymer micelle analogs exhibited rate constants for release of DOX of 0.245 and 0.278 h^− 1/2 at pH 7.4 and 5.0, respectively. These studies point to future directions to craft sophisticated devices for controlled drug release.     

Enhancement of surface ligand display on PLGA nanoparticles with amphiphilic ligand conjugates

Biodegradable polymeric nanoparticles are widely recognized as efficacious drug delivery vehicles, yet the rational engineering of nanoparticle surfaces in order to improve biodistribution, reduce clearance, and/or improve targeting remains a significant challenge. We have previously demonstrated that an amphiphilic conjugate of avidin and palmitic acid can be used to modify poly(lactic-co-glycolic acid) (PLGA) particle surfaces to display functional avidin groups, allowing for the facile attachment of biotinylated ligands for targeting or steric stabilization. Here, we hypothesized that the incorporation, density, and stability of surface-presented avidin could be modulated through varying the lipophilicity of its fatty acid conjugate partner. We tested this hypothesis by generating a set of novel conjugates incorporating avidin and common fatty acids. We found that conjugation to linoleic acid resulted in a ~ 60% increase in the incorporation of avidin on the nanoparticle surface compared to avidin-palmitic acid, which exhibited the highest avidin incorporation in previous studies. Further, the linoleic acid-avidin conjugate yielded nanoparticles with enhanced ability to bind biotinylated ligands compared to the previous method; nanoparticles modified with avidin-linoleic acid bound ~ 170% more biotin-HRP than those made with avidin-palmitic acid and ~ 1300% more than particles made without conjugated avidin. Most critically, increased ligand density on anti-CD4-targeted nanoparticles formulated with the linoleic acid-avidin conjugate resulted in a 5% increase in binding of CD4⁺ T cells. Thus we conclude that the novel avidin-linoleic acid conjugate facilitates enhanced ligand density on PLGA nanoparticles, resulting in functional enhancement of cellular targeting.     

Polyethyleneimine-based core-shell nanogels: a promising siRNA carrier for argininosuccinate synthetase mRNA knockdown in HeLa cells

RNA interference using small interfering RNA (siRNA) is a promising biological strategy for treatment of diverse diseases; however, application of siRNA is severely hindered by its poor stability and low cellular uptake efficiency. We have recently demonstrated that polyethyleneimine (PEI)-based amphiphilic core-shell particles have several distinguishing advantages over native PEI and its derivatives. This paper presents a novel type of PEI-based nanogels with a biodegradable gelatin core. The core-shell nanogels were synthesized via a two-stage reaction: (1) preparation of highly uniform gelatin nanoparticles through appropriate treatment of gelatin solution; and (2) conjugation of branched PEI to the preformed gelatin nanoparticles, followed by repeated cycles of desolvation and drying of the gelatin-PEI nanogels in ethanol/water mixture. The resulting nanogels have a well-defined nanostructure that contains a gelatin core and a PEI shell. They have an average diameter of 200 ± 40nm with high uniformity. The nanogel particles possess positive zeta-potential values of up to +40mV at neutral pH, indicating that they are highly positive and very stable in aqueous medium. The gelatin-PEI nanogels were able to completely condense siRNA at N/P ratios of as low as 5:1, and effectively protected siRNA against enzymatic degradation. Furthermore, the nanogels were four times less toxic than native PEI. Besides low toxicity, the nanogels were able to effectively deliver siRNA into HeLa cells. It was found that increasing the N/P ratio from 10 to 30 significantly increased the intracellular uptake efficiency of siRNA from 41 to 84%. Confocal laser scanning microscopic images confirmed that the nanogels were able to effectively deliver siRNA in the cytoplasm of HeLa cells. The delivered siRNA could inhibit 70% of human argininosuccinate synthetase 1 (ASS1) gene expression. This gene silencing percentage is much higher than that of the commercial Lipofectamine(TM) 2000. Our studies demonstrate that gelatin-PEI core-shell nanogels have promising potential to act as an effective siRNA carrier.     

Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue.     

Enhanced drug-loading and therapeutic efficacy of hydrotropic oligomer-conjugated glycol chitosan nanoparticles for tumor-targeted paclitaxel delivery

Enhanced drug-loading and therapeutic efficacies are highly essential properties for nanoparticles as tumor-targeting drug carriers. Herein, we developed the glycol chitosan nanoparticles with hydrotropic oligomers (HO-CNPs) as a new tumor targeting drug delivery system. For enhancing drug-loading efficiency of paclitaxel in drug carriers, hydrotropic 2-(4-(vinylbenzyloxy)-N,N-diethylnicotinamide) (VBODENA-COOH) oligomers, that were used for enhancing the aqueous solubility of paclitaxel, were directly conjugated to glycol chitosan polymers. The amphiphilic conjugates readily formed nanoparticle structure (average size = 302 ± 22 nm) in aqueous condition. Water-insoluble paclitaxel (PTX) was readily encapsulated into HO-CNPs with a high drug-loading amount up to 24.2 wt.% (2.4 fold higher than other polymeric nanoparticles) by a simple dialysis method. The PTX encapsulated HO-CNPs (PTX-HO-CNPs; average size = 343 ± 12 nm) were very stable in aqueous media up to 50 days. Also, PTX-HO-CNPs presented rapid cellular uptake and lower cytotoxicity in cell culture system, compared to Cremophor EL/ethanol formulation of PTX. In tumor-bearing mice, the extravasation and accumulation of PTX-HO-CNPs in tumor tissue were precisely observed by intravital fluorescence imaging techniques. Furthermore, PTX-HO-CNPs showed the higher therapeutic efficacy, compared to Abraxane®, a commercialized PTX-formulation. These overall results demonstrate its potential as a new nano-sized PTX carrier for cancer treatment.     

Biodegradable star HPMA polymer-drug conjugates: Biodegradability, distribution and anti-tumor efficacy

Herein, new biodegradable star polymer-doxorubicin conjugates designed for passive tumor targeting were investigated, and their synthesis, physico-chemical characterization, drug release, biodegradation, biodistribution and in vivo anti-tumor efficacy are described. In the conjugates, the core formed by poly(amidoamine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds, which enabled intracellular pH-controlled drug release. The described synthesis facilitated the preparation of biodegradable polymer conjugates in a broad range of molecular weights (200-1000 g/mol) while still maintaining low polydispersity (~ 1.7). The polymer grafts were attached to the dendrimers through either stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high-molecular-weight polymer carrier to excretable products. Biodegradability tests in suspensions of EL4 T-cell lymphoma cells showed that the rate of degradation was much faster for reductively degradable conjugates (close to completion within 24 h of incubation) than for conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (slow degradation taking several days). This finding was likely due to the differences in steric hindrance in terms of the accessibility of the small molecule glutathione and the bulky enzyme cathepsin B to the polymer substrate. Regarding drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin under mild acidic conditions that model the tumor cell microenvironment. The star polymer-Dox conjugates exhibited significantly prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice, indicating the important role of the EPR effect in its anti-cancer activity. The star polymer conjugates showed prominently higher in vivo anti-tumor activities than the free drug or linear polymer conjugate when tested in mice bearing EL4 T-cell lymphoma, with a significant number of long-term surviving (LTS). Based on the results, we conclude that a M_w of HPMA copolymers of 200,000 to 600,000 g/mol is optimal for polymer carriers designed for the efficient passive targeting to solid tumors. In addition, an expressive therapy-dependent stimulation of the immune system was observed.     

Novel micelle formulations to increase cutaneous bioavailability of azole antifungals

Efficient topical drug administration for the treatment of superficial fungal infections would deliver the therapeutic agent to the target compartment and reduce the risk of systemic side effects. However, the physicochemical properties of the commonly used azole antifungals make their formulation a considerable challenge. The objective of the present investigation was to develop aqueous micelle solutions of clotrimazole (CLZ), econazole nitrate (ECZ) and fluconazole (FLZ) using novel amphiphilic methoxy-poly(ethylene glycol)-hexyl substituted polylactide (MPEG-hexPLA) block copolymers. The CLZ, ECZ and FLZ formulations were characterized with respect to drug loading and micelle size. The optimal drug formulation was selected for skin transport studies that were performed using full thickness porcine and human skin. Penetration pathways and micellar distribution in the skin were visualized using fluorescein loaded micelles and confocal laser scanning microscopy. The hydrodynamic diameters of the azole loaded micelles were between 70 and 165 nm and the corresponding number weighted diameters (d_n) were 30 to 40 nm. Somewhat surprisingly, the lowest loading efficiency (< 20%) was observed for CLZ (the most hydrophobic of the three azoles tested); in contrast, under the same conditions, ECZ was incorporated with an efficiency of 98.3% in MPEG-dihexPLA micelles. Based on the characterization data and preliminary transport experiments, ECZ loaded MPEG-dihexPLA micelles (concentration 1.3 mg/mL; d_n < 40 nm) were selected for further study. ECZ delivery was compared to that from Pevaryl® cream (1% w/w ECZ), a marketed liposomal formulation for topical application. ECZ deposition in porcine skin following 6 h application using the MPEG-dihexPLA micelles was > 13-fold higher than that from Pevaryl® cream (22.8 ± 3.8 and 1.7 ± 0.6 μg/cm², respectively). A significant enhancement was also observed with human skin; the amounts of ECZ deposited were 11.3 ± 1.6 and 1.5 ± 0.4 μg/cm², respectively (i.e., a 7.5-fold improvement in delivery). Confocal laser scanning microscopy images supported the hypothesis that the higher delivery observed in porcine skin was due to a larger contribution of the follicular penetration pathway. In conclusion, the significant increase in ECZ skin deposition achieved using the MPEG-dihexPLA micelles demonstrates their ability to improve cutaneous drug bioavailability; this may translate into improved clinical efficacy in vivo. Moreover, these micelle systems may also enable targeting of the hair follicle and this will be investigated in future studies.     

Biophysical properties of chitosan/siRNA polyplexes: Profiling the polymer/siRNA interactions and bioactivity

Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW = 44, 63, 93 and 143 kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4-8; N:P) similar positive zeta potential (20-30 mV) and comparable particle sizes (about 150 nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5-150 nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150 nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta H_bind(− 84 to −102 kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44 kDa derived polyplexes exhibited regular spherical structure, whereas the 143 kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar bioactivity.     

Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer

For efficient cancer therapy, small interfering RNA (siRNA) should be stably and efficiently delivered into the target tissue and readily taken up by cancer cells. To address these needs, a polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy. The interaction between poly(ethylene glycol) (PEG)-conjugated vascular endothelial growth factor siRNA (VEGF siRNA–PEG) and polyethylenimine (PEI) led to the spontaneous formation of nanoscale polyelectrolyte complex micelles (VEGF siRNA–PEG/PEI PEC micelles), having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous as well as intratumoral administration of the PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal tumor model without showing any detectable inflammatory responses in mice. Upon examination of the PEC micelle distribution and in vivo optical imaging following intravenously injection, enhanced accumulation of the PEC micelles was also observed in the tumor region. This study demonstrates the feasibility of using PEC micelles as a potential carrier for therapeutic siRNAs in local and systemic treatment of cancer.     

The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low M_w free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2–4 mM octreotide or leuprolide acetate salts in a 0.1 M HEPES buffer, pH 7.4, with polymer particles or films at 4–37 °C for 24 h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy, stimulated Raman scattering (SRS) and laser scanning confocal imaging, and microtome sectioning techniques were used to examine peptide penetration into the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST + 0.02% sodium azide, 37 °C) and in vivo (male Sprague–Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but also can be internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~ 17 wt.% leuprolide loading in the polymer (i.e., ~ 70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for > 2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague–Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides.     

Rational design of biodegradable cationic polycarbonates for gene delivery

Polycarbonates provide an attractive option for use as gene delivery vectors owing to their biocompatibility and ease of incorporating functional moieties. In this study, we described an approach to synthesize cationic polymers with well-defined molecular weights and narrow polydispersities by an organocatalytic ring-opening polymerization of functional cyclic carbonates containing alkyl halide side chains, followed by a subsequent functionalization step with bis-tertiary amines designed to facilitate gene binding and endosomal escape. The cationic polycarbonate effectively condensed DNA at low N/P ratios, generating nanoparticles (83 to 124 nm in diameter) with positive zeta potentials (~ 27 mV). In addition, reporter gene expression efficiencies in HepG2, HEK293, MCF-7 and 4T1 cell lines were high even in the presence of serum. Importantly, the polycarbonate delivery agent demonstrated minimal cytotoxicity at the optimal N/P ratios determined to confer high gene expression efficiencies. Therefore, this biodegradable polymer is presented as a promising non-viral vector for gene delivery.     

DPD simulations on morphologies and structures of blank PLGA-b-PEG-b-PLGA polymeric micelles and docetaxel-loaded PLGA-b-PEG-b-PLGA polymeric micelles

Dissipative particle dynamics (DPD) simulation was used to study the morphologies and structures of blank (no drug) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-b-PEG-b-PLGA) polymeric micelles and the docetaxel (Dtx)-loaded PLGA-b-PEG-b-PLGA polymeric micelles. We focused on the influences of PLGA-b-PEG-b-PLGA copolymer concentration, composition, Dtx drug content and the shear rate on morphologies and structures of the micelles. Our simulations show that the PLGA-b-PEG-b-PLGA copolymers in the aqueous solutions could aggregate and form blank micelles while Dtx drug and PLGA-b-PEG-b-PLGA could aggregate and form drug-loaded micelles. Under different PLGA-b-PEG-b-PLGA concentrations and drug content, the blank and drug-loaded micelles are observed as spherical, onionlike, columnar, and lamellar structures. The onionlike structures are comprised of the PEG hydrophilic core, the PLGA hydrophobic middle layer, and the PEG hydrophilic shell. As the structure of micelles varies from a spherical core–shell structure to a core–middle layer–shell onionlike structure, the distribution of the Dtx drugs diffuses from the core to the PLGA middle layer of the aggregate. In addition, the drug release process of the Dtx-loaded micelles under shear flow is also simulated. And the results show that the spherical micelles turn into a columnar structure under a shear rate from 0.2 to 3.4. When the shear rate increases to 3.5, the Dtx drugs released gradually increase until all are released with time evolution. These findings illustrate the dependence of the structural morphologies on the detailed molecular parameters of PLGA-b-PEG-b-PLGA and Dtx.

Dissipative particle dynamics simulation was used to study the morphologies and structures of blank (no drug) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) polymeric micelles and the docetaxel-loaded polymeric micelles.     

A duplex oligodeoxynucleotide-dendrimer bioconjugate as a novel delivery vehicle for doxorubicin in in vivo cancer therapy

We designed a bioconjugate between duplex oligodeoxynucleotides (dODNs) and a dendrimer (DEN) and demonstrate its feasibility as a novel delivery system for doxorubicin (Dox) in animal tumor models and against cancer cells in vitro. The dODNs-DEN conjugates formed stable complexes with Dox (~ 184 Dox molecules per conjugate) and the resulting Dox-loaded conjugate exhibited a sustained drug release pattern both in vitro and in vivo. Pharmacokinetic studies showed that Dox-loaded dODNs-DEN conjugates were cleared from plasma much more slowly (up to 5.3 h) than was free Dox (0.65 h). Furthermore, tumors retained a higher amount of Dox in mice treated with the conjugate group compared to that of free Dox-treated group at the same dosage. In mice bearing 4T1 murine breast tumor allografts, the dendrimer conjugate, at a Dox concentration of 1 mg/kg, was more effective than the equivalent concentration of free Dox and tumor size reduction was equivalent to that seen using 4 mg/kg free Dox. We observed no severe systemic toxicity or cardiotoxicity in mice treated with the conjugate, as indicated by body weight change and heart tissue histology. These findings indicate that dODNs-DEN conjugates can be used to administer Dox with improved pharmacokinetics, lower toxicity, and an increased ability to concentrate drugs in tumors, compared with free drug, and that such conjugates are effective against tumors in vivo.     

Tumor pH-responsive flower-like micelles of poly(l-lactic acid)-b-poly(ethylene glycol)-b-poly(l-histidine)

Polymeric micelles were constructed from poly(l-lactic acid) (PLA; M_n 3K)-b-poly(ethylene glycol) (PEG; M_n 2K)-b-poly(l-histidine) (polyHis; M_n 5K) as a tumor pH-specific anticancer drug carrier. Micelles (particle diameter: ∼ 80 nm; critical micelle concentration (CMC): 2 μg/ml) formed by dialysis of the polymer solution in dimethylsulfoxide (DMSO) against pH 8.0 aqueous solution, are assumed to have a flower-like assembly of PLA and polyHis blocks in the core and PEG block as the shell. The pH-sensitivity of the micelles originates from the deformation of the micellar core due to the ionization of polyHis at a slightly acidic pH. However, the co-presence of pH-insensitive lipophilic PLA block in the core prevented disintegration of the micelles and caused swelling/aggregation. A fluorescence probe study showed that the polarity of pyrene retained in the micelles increased as pH was decreased from 7.4 to 6.6, indicating a change to a more hydrophilic environment in the micelles. Considering that the size increased up to 580 nm at pH 6.6 from 80 nm at pH 7.4 and that the transmittance of micellar solution increased with decreasing pH, the micelles were not dissociated but rather swollen/aggregated. Interestingly, the subsequent decline of pyrene polarity below pH 6.6 suggested re-self-assembly of the block copolymers, most likely forming a PLA block core while polyHis block relocation to the surface. Consequently, these pH-dependent physical changes of the PLA-b-PEG-b-polyHis micelles provide a mechanism for triggered drug release from the micelles triggered by the small change in pH (pH 7.2–6.5).