The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short-or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides.
GM1; hGLP-1; drug delivery; transcytosis
Aseptic implant loosening related to implant wear particle-induced inflammation is the most common cause of failure after joint replacement. Modulation of the inflammatory reaction to the wear products represents a rational approach for preventing aseptic implant failure. Long-term treatment using anti-inflammatory agents, however, can be associated with significant systemic side effects due to the drugs' lack of tissue specificity. To address this issue, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-dexamethasone conjugate (P-Dex) was developed and evaluated for prevention of wear particle-induced osteolysis and the loss of fixation in a murine prosthesis failure model. Daily administration of free dexamethasone (Dex) was able to prevent wear particle-induced osteolysis, as assessed by micro-CT and histological analysis. Remarkably, monthly P-Dex administration (dose equivalent to free Dex treatment) was equally effective as free dexamethasone, but was not associated with systemic bone loss (a major adverse side effect of glucocorticoids). The reduced systemic toxicity of P-Dex is related to preferential targeting of the sites of wear particle-induced inflammation and its subcellular sequestration and retention by local inflammatory cell populations, resulting in sustained therapeutic action. These results demonstrate the feasibility of utilizing a macromolecular prodrug with reduced systemic toxicity to prevent wear particle-induced osteolysis.
HPMA copolymer; prodrug; inflammation targeting; dexamethasone; implant loosening; ELVIS
The accessibility of extravascular tumor tissue to drugs is critical for therapeutic efficacy. We previously described a tumor-targeting peptide (iRGD) that elicits active transport of drugs and macromolecules (covalently coupled or co-administered) across the vascular wall into tumor tissue. Short peptides (iRGD is a 9-amino acid cyclic peptide) generally have a plasma half-life measured in minutes. Since short half-life limits the window of activity obtained with a bolus injection of iRGD, we explored to extend the half-life of the peptide. We show here that addition of a cysteine residue prolongs the plasma half-life of iRGD and increases the accumulation of the peptide in tumors. This modification prolongs the activity of iRGD in inducing macromolecular extravasation and leads to greater drug accumulation in tumors than is obtained with the unmodified peptide. This effect is mediated by covalent binding of iRGD to plasma albumin through a disulfide bond. Our study provides a simple strategy to improve peptide pharmacokinetics and activity. Applied to RGD, it provides a means to increase the entry of therapeutic agents into tumors.
iRGD; extra cysteine; half-life; tumor extravasation; bystander activity; albumin
Enhanced in vivo gene expression using non-viral vectors is a critical issue in gene therapy in general. Among the many potential utilities of non-viral vector-mediated gene delivery, its application in DNA-based vaccination is an attractive approach with several practical advantages over conventional vaccination. We have previously shown that the endosomolytic bacterial protein listeriolysin O (LLO) is capable of facilitating transfection in vitro using the LPDII (anionic liposome-polycation-DNA complexes) delivery system. In the present study we have extended and investigated the DNA delivery of LLO-containing LPDII to in vivo and evaluated its utility in DNA vaccination in mice. We further investigated the ability of this non-viral gene delivery system to elicit an immune response to a model antigen ovalbumin (OVA), particularly focusing on the OVA-specific CD8+ cytotoxic T lymphocyte (CTL) response, after delivery of a plasmid containing the OVA cDNA. A DNA prime and protein boost protocol was employed to generate cytotoxic T cell responses. Our results show that increased in vitro and in vivo transfection efficiencies were observed when LLO was incorporated into LPDII. This LLO-LPDII formulation produced an enhanced functional antigen-specific CD8+ T cell response in vivo compared to the heat-inactivated LLO-containing LPDII (HI-LLO-LPDII) formulation. Furthermore, a significantly higher CTL frequency was observed in the splenocytes isolated from the mice primed with LLO-LPDII by an enzyme-linked immunosorbent spot assay. Interferon-γ production upon specific stimulation by OVA-specific CD8+ peptide was also significantly stronger with the inclusion of LLO into LPDII. These findings suggest that the LLO-containing LPDII system possesses noteworthy potential as a candidate carrier for DNA vaccine delivery.
listeriolysin O; LPDII; DNA delivery; vaccine; CTL response
Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB.
By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.
Phage display screening; peptides; nanocarriers; liposomes; targeted therapy; neuroblastoma
The poor solubility of cisplatin (CDDP) often presents a major obstacle in the formulation of CDDP in nanoparticles (NPs) by traditional methods. We have developed a novel method for synthesizing CDDP NPs taking advantage of its poor solubility. By mixing two reverse microemulsions containing KCl and a highly soluble precursor of CDDP, cis-diaminedihydroplatinum (II), we have successfully formulated CDDP NPs with a controllable size (in the range of 12–75 nm) and high drug loading capacity (approximately 80 wt%). The formulation was done in two steps. The pure CDDP NPs were first stabilized for dispersion in an organic solvent by coating with 1, 2-dioleoyl-sn-glycero-3-phosphate (DOPA). Both x-ray photoelectron spectroscopy and 1H NMR data confirmed that the major ingredient of the DOPA-coated NPs was CDDP. After purification, additional lipids were added to stabilize the NPs for dispersion in an aqueous solution. The final NPs contain a lipid bilayer coating and are named Lipid-Pt-Cl (LPC) NPs, which showed significant antitumor activity both in vitro and in vivo. Thus, CDDP precipitate serves as the major material for assembling the novel NPs. This unique method of nanoparticle synthesis may be applicable in formulating other insoluble drugs.
Cisplatin; Nanoparticle; Drug delivery; Nanoprecipitate; Microemulsion
Liposomes; nanotechnology; topotecan; camptothecins; kinetics; membrane binding; permeability
Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid; into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, OA containing LNPs delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, OA containing LNPs showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. OA containing LNPs are a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases.
Cationic lipid nanoparticles; Helper lipids; siRNA; microRNA; Hepatic delivery
While potent cytotoxic agents are available to oncologists, the clinical utility of these agents is limited due to their non-specific distribution in the body and toxicity to normal tissues leading to use of suboptimal doses for eradication of metastatic disease. Furthermore, treatment of micrometastases is impeded by several biobarriers, including their small size and high dispersion to organs, making them nearly inaccessible to drugs. To circumvent these limitations in treating metastatic disease, we developed a multicomponent, flexible chain-like nanoparticle (termed nanochain) that possesses a unique ability to gain access to and be deposited at micrometastatic sites. Moreover, coupling nanochain particles to radiofrequency (RF)-triggered cargo delivery facilitated widespread delivery of drug into hard-to-reach cancer cells. Collectively, these features synergistically facilitate effective treatment and ultimately eradication of micrometastatic disease using a low dose of a cytotoxic drug.
Chain-like nanoparticle; targeting; cancer metastasis; nanochains; radiofrequency-triggered drug release
Aluminum hydroxide is used as a vaccine adjuvant in various human vaccines. Unfortunately, despite its favorable safety profile, aluminum hydroxide can only weakly or moderately potentiate antigen-specific antibody responses. When dispersed in an aqueous solution, aluminum hydroxide forms particulates of 1–20 µm. There is increasing evidence that nanoparticles around or less than 200 nm as vaccine or antigen carriers have a more potent adjuvant activity than large microparticles. In the present study, we synthesized aluminum hydroxide nanoparticles of 112 nm. Using ovalbumin and Bacillus anthracis protective antigen protein as model antigens, we showed that protein antigens adsorbed on the aluminum hydroxide nanoparticles induced a stronger antigen-specific antibody response than the same protein antigens adsorbed on the traditional aluminum hydroxide microparticles of around 9.3 µm. The potent adjuvant activity of the aluminum hydroxide nanoparticles was likely related to their ability to more effectively facilitate the uptake of the antigens adsorbed on them by antigen-presenting cells. Finally, the local inflammation induced by aluminum hydroxide nanoparticles in the injection sites was milder than that induced by microparticles. Simply reducing the particle size of the traditional aluminum hydroxide adjuvant into nanometers represents a novel and effective approach to improve its adjuvanticity.
Vaccine; nanoparticles; microparticles; antibody responses; local inflammation
Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into skin.
Topical; Hyaluronan; healing; wound; repair; wrinkle
Previously we have developed and statistically validated Quantitative Structure Property Relationship (QSPR) models that correlate drugs’ structural, physical and chemical properties as well as experimental conditions with the relative efficiency of remote loading of drugs into liposomes (Cern et al, Journal of Controlled Release, 160(2012) 14–157). Herein, these models have been used to virtually screen a large drug database to identify novel candidate molecules for liposomal drug delivery. Computational hits were considered for experimental validation based on their predicted remote loading efficiency as well as additional considerations such as availability, recommended dose and relevance to the disease. Three compounds were selected for experimental testing which were confirmed to be correctly classified by our previously reported QSPR models developed with Iterative Stochastic Elimination (ISE) and k-nearest neighbors (kNN) approaches. In addition, 10 new molecules with known liposome remote loading efficiency that were not used in QSPR model development were identified in the published literature and employed as an additional model validation set. The external accuracy of the models was found to be as high as 82% or 92%, depending on the model. This study presents the first successful application of QSPR models for the computer-model-driven design of liposomal drugs.
Liposomes; Remote loading; QSPR; Virtual screening; Iterative Stochastic Elimination, k-nearest neighbors
The objective of a systemically administered cancer gene therapy is to achieve gene expression that is isolated to the tumor tissue. Unfortunately, viral systems have strong affinity for the liver, and delivery from non-viral cationic systems often results in high expression in the lungs. Non-specific delivery to these organs must be overcome if tumors are to be aggressively treated with genes such as IL-12 which activates a tumor immune response, and TNF-alpha which can induce tumor cell apoptosis. Techniques which have led to specific expression in tumor tissue include receptor targeting through ligand conjugation, utilization of tumor specific promoters and viral mutation in order to take advantage of proteins overexpressed in tumor cells. This review analyzes these techniques applied to liposomal, PEI, dendrimer, stem cell and viral gene delivery systems in order to determine the techniques that are most effective in achieving tumor specific gene expression after systemic administration.
Gene Therapy; Systemic; Viral; Non-Viral; Dendrimer; Delivery System
Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber’s Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65−/− model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65−/− mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy.
RPE65; non-viral gene therapy; retinal pigment epithelium; DNA nanoparticle; S/MAR
Current treatments for prostate cancer are still not satisfactory, often resulting in tumor regrowth and metastasis. One of the main reasons for the ineffective anti-prostate cancer treatments is the failure to deplete cancer stem-like cells (CSCs) - a subset of cancer cells with enhanced tumorigenic capacity. Thus, combination of agents against both CSCs and bulk tumor cells may offer better therapeutic benefits. Several molecules with anti-cancer stem/progenitor cell activities have been under preclinical evaluations. However, their low solubility and nonspecific toxicity limit their clinical translation. Herein, we designed a combination macromolecular therapy containing two drug conjugates: HPMA copolymer-cyclopamine conjugate (P-CYP) preferentially toxic to cancer stem/progenitor cells, and HPMA copolymer-docetaxel conjugate (P-DTX) effective in debulking the tumor mass. Both conjugates were synthesized using RAFT (reversible addition-fragmentation chain transfer) polymerization resulting in narrow molecular weight distribution. The killing effect of the two conjugates against bulk tumor cells and CSCs were evaluated in vitro and in vivo. In PC-3 or RC-92a/hTERT prostate cancer cells, P-CYP preferentially kills and impairs the function of CD133+ prostate cancer stem/progenitor cells; P-DTX was able to kill bulk tumor cells instead of CSCs. In PC-3 xenograft mice model, combination of P-DTX and P-CYP showed the most effective and persistent tumor growth inhibitory effect. In addition, residual tumors contained less CD133+ cancer cells following combination or P-CYP treatments, indicating selective killing of cancer cells with stem/progenitor cell properties.
N-(2-hydroxypropyl)methacrylamide (HPMA); Combination therapy; Cancer stem/progenitor cells; Cyclopamine; Docetaxel
Biodegradable polymer microparticles are promising delivery depots for protein therapeutics due to their relatively simple fabrication and facile administration. Double-wall microspheres (DWMS) comprising a core and shell made of two distinct polymers may provide enhanced control of the drug release profiles. Using precision particle fabrication (PPF) technology, monodisperse DWMS were fabricated with model protein bovine serum albumin (BSA)-loaded poly(lactide-co-glycolide) (PLG) core and drug-free poly(d,l-lactic acid) (PDLL) shell of uniform thickness. Monolithic single-wall microspheres were also fabricated to mimic the BSA-loaded PLG core. Using ethyl acetate and dichloromethane as shell- and core-phase solvents, respectively, BSA was encapsulated selectively in the core region within DWMS with higher loading and encapsulation efficiency compared to using dichloromethane as core and shell solvents. BSA in vitro release rates were retarded by the presence of the drug-free PDLL shell. Moreover, increasing PDLL shell thickness resulted in decreasing BSA release rate. With a 14-µm thick PDLL shell, an extended period of constant-rate release was achieved.
Monodisperse double-wall microspheres; Poly(lactide-co-glycolide); Poly(lactic acid); Bovine serum albumin; Controlled release
Traditional antibiotic therapy to control medical device-based infections typically fails to clear biofilm infections and may even promote the evolution of antibiotic resistant species. We report here the development of two novel antibiofilm agents; gallium (Ga) or zinc (Zn) complexed with protoporphyrin IX (PP) or mesoprotoporphyrin IX (MP) that are both highly effective in negating suspended bacterial growth and biofilm formation. These chelated gallium or zinc complexes act as iron siderophore analogs, surplanting the natural iron uptake of most bacteria. Poly (ether urethane) (PEU; Biospan®) polymer films were fabricated for the controlled sustained release of the Ga- or Zn-complexes, using an incorporated pore-forming agent, poly (ethylene glycol) (PEG). An optimum formulation containing 8% PEG (MW=1450) in the PEU polymer effectively sustained drug release for at least 3 months. All drug-loaded PEU films exhibited in vitro ≥ 90% reduction of Gram-positive (Staphylococcus epidermidis) and Gram-negative (Pseudomonas aeruginosa) bacteria in both suspended and biofilm culture versus the negative control PEU films releasing nothing. Cytotoxicity and endotoxin evaluation demonstrated no adverse responses to the Ga- or Zn-complex releasing PEU films. Finally, in vivo studies further substantiate the anti-biofilm efficacy of the PEU films releasing Ga- or Zn- complexes.
Anti-biofilm biomaterials; interrupting iron metabolism, gallium and zinc siderophores, poly (ether urethane); drug release; Staphylococcus epidermidis; Pseudomonas aeruginosa
Multidrug resistance (MDR) is a significant problem in the clinical management of several cancers. Overcoming MDR generally involves multi-modal therapeutic approaches that integrates enhancement of delivery efficiency using targeted nano-platforms as well as strategies that can sensitize cancer cells to drug treatments. We recently demonstrated that tandem delivery of siRNAs that downregulate anti-apoptotic genes overexpressed in cisplatin resistant tumors followed by therapeutic challenge using cisplatin loaded in CD44 targeted hyaluronic acid (HA) nanoparticles (NPs) induced synergistic antitumor response in CD44 expressing tumors that are resistant to cisplatin. In the current study, a near infrared (NIR) dye-loaded HA NPs was employed to image the whole body localization of NPs after intravenous (i.v.) injection into live mice bearing human lung tumors that were sensitive and resistant to cisplatin. In addition, we quantified the siRNA duplexes and cisplatin dose distribution in various tissues and organs using an ultra-sensitive quantitative PCR method and inductively coupled plasma-mass spectrometry (ICP-MS), respectively, after i.v. injection of the payload loaded HA NPs in tumor bearing mice. Our findings demonstrate that the distribution pattern of the siRNA and cisplatin using specifically engineered CD44 targeting HA NPs correlated well with the tumor targeting capability as well as the activity and efficacy obtained with combination treatments.
Hyaluronic acid; siRNA; cisplatin; nanoparticles; tumor targeting; combination therapy; multidrug resistance
Although the careful selection of shell-forming polymers for the construction of nanoparticles is an obvious parameter to consider for shielding of core materials and their payloads, providing for prolonged circulation in vivo by limiting uptake by the immune organs, and thus, allowing accumulation at the target sites, the immunotoxicities that such shielding layers elicit is often overlooked. For instance, we have previously performed rigorous in vitro and in vivo comparisons between two sets of nanoparticles coated with either non-ionic poly(ethylene glycol) (PEG) or zwitterionic poly(carboxybetaine) (PCB), but only now report the immunotoxicity and anti-biofouling properties of both polymers, as homopolymers or nanoparticle-decorating shell, in comparison to the uncoated nanoparticles, and Cremophor-EL, a well-known low molecular weight surfactant used for formulation of several drugs. It was found that both PEG and PCB polymers could induce the expression of cytokines in vitro and in vivo, with PCB being more immunotoxic than PEG, which corroborates the in vivo pharmacokineties and biodistribution profiles of the two sets of nanoparticles. This is the first study to report on the ability of PEG, the most commonly utilized polymer to coat nanomaterials, and PCB, an emerging zwitterionic anti-biofouling polymer, to induce the secretion of cytokines and be of potential immunotoxicity. Furthermore, we report here on the possible use of immunotoxicity assays to partially predict in vivo pharmacokineties and biodistribution of nanomaterials.
Poly(ethylene glycol); poly(carboxybetaine); Cremophor-EL; protein adsorption; stealth; antibiofouling; immunotoxicity; cytokines; nanoparticles; pharmacokineties; biodistribution
Focused ultrasound (FUS) in the presence of systemically administered microbubbles has been shown to locally, transiently and reversibly increase the permeability of the blood-brain barrier (BBB), thus allowing targeted delivery of therapeutic agents in the brain for the treatment of central nervous system diseases. Currently, microbubbles are the only agents that have been used to facilitate the FUS-induced BBB opening. However, they are constrained within the intravascular space due to their micron-size diameters, limiting the delivery effect at or near the microvessels. In the present study, acoustically-activated nanodroplets were used as a new class of contrast agents to mediate FUS-induced BBB opening in order to study the feasibility of utilizing these nanoscale phase-shift particles for targeted drug delivery in the brain. Significant dextran delivery was achieved in the mouse hippocampus using nanodroplets at clinically relevant pressures. Passive cavitation detection was used in the attempt to establish a correlation between the amount of dextran delivered in the brain and the acoustic emission recorded during sonication. Conventional microbubbles with the same lipid shell composition and perfluorobutane core as the nanodroplets were also used to compare the efficiency of FUS-induced dextran delivery. It was found that nanodroplets had a higher BBB opening pressure threshold but a lower stable cavitation threshold than microbubbles, suggesting that contrast agent-dependent acoustic emission monitoring was needed. More homogeneous dextran delivery within the targeted hippocampus was achieved using nanodroplets without inducing inertial cavitation or compromising safety. Our results offered a new means of developing the FUS-induced BBB opening technology for potential extravascular targeted drug delivery in the brain, extending the potential drug delivery region beyond the cerebral vasculature.
Doxorubicin (DXR) and daunorubicin (DNR) inhibit hypoxia-inducible factor-1 (HIF-1) transcriptional activity by blocking its binding to DNA. Intraocular injections of DXR or DNR suppressed choroidal and retinal neovascularization (NV), but also perturbed retinal function as demonstrated by electroretinograms (ERGs). DXR was conjugated to novel copolymers of branched polyethylene glycol and poly(sebacic acid) (DXR-PSA-PEG3) and formulated into nanoparticles that when placed in aqueous buffer, slowly released small DXR-conjugates. Intraocular injection of DXR-PSA-PEG3 nanoparticles (1 or 10 μg DXR content) reduced HIF-1-responsive gene products, strongly suppressed choroidal and retinal NV, and did not cause retinal toxicity. In transgenic mice that express VEGF in photoreceptors, intraocular injection of DXR-PSA-PEG3 nanoparticles (10 μg DXR content) suppressed NV for at least 35 days. Intraocular injection of DXR-PSA-PEG3 nanoparticles (2.7 mg DXR content) in rabbits resulted in sustained DXR-conjugate release with detectable levels in aqueous humor and vitreous for at least 105 days. This study demonstrates a novel HIF-1-inhibitor-polymer conjugate formulated into controlled-release particles that maximizes efficacy and duration of activity, minimizes toxicity, and provides a promising new chemical entity for treatment of ocular NV.
age-related macular degeneration; angiogenesis; diabetic retinopathy; nanoparticles
Intraperitoneal therapy (IP) has demonstrated survival advantages in patients with peritoneal cancers, but has not become a widely practiced standard-of-care in part due to local toxicity and sub-optimal drug delivery. Paclitaxel-loaded, polymeric microparticles were developed to overcome these limitations. The present study evaluated the effects of microparticle properties on paclitaxel release (extent and rate) and in vivo pharmacodynamics. In vitro paclitaxel release from microparticles with varying physical characteristics (i.e., particle size, copolymer viscosity and composition) was evaluated. A method was developed to simulate the dosing rate and cumulative dose released in the peritoneal cavity based on the in vitro release data. The relationship between the simulated drug delivery and treatment outcomes of seven microparticle compositions was studied in mice bearing IP human pancreatic tumors, and compared to that of the intravenous Cremophor micellar paclitaxel solution used off-label in previous IP studies. Paclitaxel release from polymeric microparticles in vitro was multi-phasic; release was greater and more rapid from microparticles with lower polymer viscosities and smaller diameters (e.g., viscosity of 0.17 vs. 0.67 dl/g and diameter of 5–6 vs. 50–60 μm). The simulated drug release in the peritoneal cavity linearly correlated with treatment efficacy in mice (r2>0.8, p<0.001). The smaller microparticles, which distribute more evenly in the peritoneal cavity compared to the large microparticles, showed greater dose efficiency. For single treatment, the microparticles demonstrated up to 2-times longer survival extension and 4-times higher dose efficiency, relative to the paclitaxel/Cremophor micellar solution. Upon repeated dosing, the paclitaxel/Cremophor micellar solution showed cumulative toxicity whereas the microparticle that yielded 2-times longer survival did not display cumulative toxicity. The efficacy of IP therapy depended on both temporal and spatial factors that were determined by the characteristics of the drug delivery system. A combination of fast- and slow-releasing microparticles with 5–6 μm diameter provided favorable spatial distribution and optimal drug release for IP therapy.
Intraperitoneal therapy; paclitaxel; PLGA microparticles; controlled release; in vitro-in vivo correlation
An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw 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 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 and stimulated Raman scattering (SRS) and laser scanning confocal imaging techniques were used to examine peptide penetration in 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 can also 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.
peptide; sorption; PLGA; kinetics; microencapsulation; controlled release