Jayanth Panyam

BIODEGRADABLE NANOPARTICLES FOR DRUG AND GENE DELIVERY TO CELLS AND TISSUE

Biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted/localized delivery of different agents including plasmid DNA, proteins and peptides and low molecular weight compounds. Research about the mechanism of intracellular uptake of nanoparticles, their trafficking and sorting into different intracellular compartments, and the mechanism of enhanced therapeutic efficacy of nanoparticle-encapsulated agent at cellular level is more recent and is the primary focus of the review. Recent studies in our laboratory demonstrated rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytosol following their uptake. Based on the above mechanism, various potential applications of nanoparticles for delivery of therapeutic agents to the cells and tissue are discussed.

Rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery

The endo‐lysosomal escape of drug carriers is crucial to enhancing the efficacy of their macromolecular payload, especially the payloads that are susceptible to lysosomal degradation. Current vectors that enable the endo‐lysosomal escape of macromolecules such as DNA are limited by their toxicity and by their ability to carry only limited classes of therapeutic agents. In this paper, we report the rapid (<10 min) endo‐lysosomal escape of biodegradable nanoparticles (NPs) formulated from the copolymers of poly(DLlactide‐co‐glycolide) (PLGA). The mechanism of rapid escape is by selective reversal of the surface charge of NPs (from anionic to cationic) in the acidic endolysosomal compartment, which causes the NPs to interact with the endo‐lysosomal membrane and escape into the cytosol. PLGA NPs are able to deliver a variety of therapeutic agents, including macromolecules such as DNA and low molecular weight drugs such as dexamethasone, intracellularly at a slow rate, which results in a sustained therapeutic effect. PLGA has a number of advantages over other polymers used in drug and gene delivery including biodegradability, biocompatibility, and approval for human use granted by the U.S. Food and Drug Administration. Hence PLGA is well suited for sustained intracellular delivery of macromolecules.—Panyam, J., Zhou, W. Z., Prabha, S., Sahoo, S. K., Labhasetwar, V. Rapid endo‐lysosomal escape of poly(DL‐lactide‐co‐glycolide) nanoparticles: implications for drug and gene delivery. FASEB J. 16, 1217–1226 (2002)

Residual polyvinyl alcohol associated with poly (d,l-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake

Polyvinyl alcohol (PVA) is the most commonly used emulsifier in the formulation of poly lactide and poly (D,L-lactide-co-glycolide) (PLGA) polymeric nanoparticles. A fraction of PVA remains associated with the nanoparticles despite repeated washing because PVA forms an interconnected network with the polymer at the interface. The objective of this study was to determine the parameters that influence the amount of residual PVA associated with PLGA nanoparticles and its effect on the physical properties and cellular uptake of nanoparticles. Nanoparticles were formulated by a multiple emulsion-solvent evaporation technique using bovine serum albumin (BSA) as a model protein. The parameters that affected the amount of residual PVA include the concentration of PVA and the type of organic solvent used in the emulsion. The residual PVA, in turn, influenced different pharmaceutical properties of nanoparticles such as particle size, zeta potential, polydispersity index, surface hydrophobicity, protein loading and also slightly influenced the in vitro release of the encapsulated protein. Importantly, nanoparticles with higher amount of residual PVA had relatively lower cellular uptake despite their smaller particle size. It is proposed that the lower intracellular uptake of nanoparticles with higher amount of residual PVA could be related to the higher hydrophilicity of the nanoparticle surface. In conclusion, the residual PVA associated with nanoparticles is an important formulation parameter that can be used to modulate the pharmaceutical properties of PLGA nanoparticles.

Size-dependency of nanoparticle-mediated gene transfection: Studies with fractionated nanoparticles

Nanoparticles formulated from biodegradable polymers such as poly (lactic acid) and poly (D,L-lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their sustained release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated using an emulsion-solvent evaporation technique. However, this formulation procedure results in the formation of particles with heterogeneous size distribution. The objective of the present study was to determine the relative transfectivity of the smaller- and the larger-sized fractions of nanoparticles in cell culture. PLGA nanoparticles containing a plasmid DNA encoding luciferase protein as a marker were formulated by a multiple emulsion-solvent evaporation method (mean particle diameter = 97 +/- 3 nm) and were fractionated using a membrane (pore size: 100 nm) filtration technique. The particles that passed through the membrane were designated as the smaller-sized nanoparticles (mean diameter = 70 +/- 2 nm) and the fraction that was retained on the membrane as the larger-sized nanoparticles (mean diameter = 202 +/- 9 nm). The smaller-sized nanoparticles showed a 27-fold higher transfection than the larger-sized nanoparticles in COS-7 cell line and a 4-fold higher transfection in HEK-293 cell line. The surface charge (zeta potential), cellular uptake, and the DNA release were almost similar for the two fractions of nanoparticles, suggesting that some other yet unknown factor(s) is responsible for the observed differences in the transfection levels. The results suggest that the particle size is an important factor, and that the smaller-sized fraction of the nanoparticle formulation predominantly contributes towards their transfection.

Polymer degradation and in vitro release of a model protein from poly(D,L-lactide-co-glycolide) nano- and microparticles

The objective of the study was to investigate the effect of particle size of nano- and microparticles formulated from poly(D,L-lactide-co-glycolide) (50:50 PLGA) on polymer degradation and protein release. Since the surface area to volume ratio is inversely proportional to the particle size, it is hypothesized that the particle size would influence the polymer degradation as well as the release of the encapsulated protein. PLGA nano- and microparticles of approximate mean diameters of 0.1, 1 and 10 microm, containing bovine serum albumin as a model protein, were formulated using a multiple water-in-oil-in-water emulsion solvent evaporation technique. These particles were incubated at 37 degrees C in phosphate-buffered saline (pH 7.4, 154 mM) and the particles were characterized at various time points for molecular weight of polymer, surface-associated polyvinyl alcohol content (PVA), and the particle surface topology using scanning electron microscopy. The supernatants from the above study were analyzed for the released protein and PVA content. Polymer degradation was found to be biphasic in both nano- and microparticles, with an initial rapid degradation for 20-30 days followed by a slower degradation phase. The 0.1 microm diameter nanoparticles demonstrated relatively higher polymer degradation rate (P<0.05) during the initial phase as compared to the larger size microparticles (first order degradation rate constants of 0.028 day(-1), 0.011 day(-1) and 0.018 day(-1) for 0.1, 1 and 10 microm particles, respectively), however the degradation rates were almost similar (0.008 to 0.009 day(-1)) for all size particles during the later phase. All size particles maintained their structural integrity during the initial degradation phase; however, this was followed by pore formation, deformation and fusion of particles during the slow degradation phase. Protein release from 0.1 and 1 microm particles was greater than that from 10 microm size particles. In conclusion, the polymer degradation rates in vitro were not substantially different for different size particles despite a 10- and 100-fold greater surface area to volume ratio for 0.1 microm size nanoparticles as compared to 1 and 10 microm size microparticles, respectively. Relatively higher amounts of the surface-associated PVA found in the smaller-size nanoparticles (0.1 microm) as compared to the larger-size microparticles could explain some of the observed degradation results with different size particles.

Dynamics of Endocytosis and Exocytosis of Poly(D,L-Lactide-co-Glycolide) Nanoparticles in Vascular Smooth Muscle Cells

AbstractPurpose. The purpose of this work was to characterize the process of endocytosis, exocytosis, and intracellular retention of poly (D,L-lactide-co-glycolide) nanoparticles in vitro using human arterial vascular smooth muscle cells (VSMCs).nMethods. Nanoparticles containing bovine serum albumin (BSA) as a model protein and 6-coumarin as a fluorescent marker were formulated by a double emulsion-solvent evaporation technique. The endocytosis and exocytosis of nanoparticles in VSMCs were studied using confocal microscopy and their intracellular uptake and retention were determined quantitatively using high-performance liquid chromatography.nResults. Cellular uptake of nanoparticles (mean particle size 97 ± 3 nm) was a concentration-, time-, and energy-dependent endocytic process. Confocal microscopy demonstrated that nanoparticles were internalized rapidly, with nanoparticles seen inside the cells as early as within 1 min after incubation. The nanoparticle uptake increased with incubation time in the presence of nanoparticles in the medium; however, once the extracellular nanoparticle concentration gradient was removed, exocytosis of nanoparticles occurred with about 65% of the internalized fraction undergoing exocytosis in 30 min. Exocytosis of nanoparticles was slower than the exocytosis of a fluid phase marker, Lucifer yellow. Furthermore, the exocytosis of nanoparticles was reduced after the treatment of cells with the combination of sodium azide and deoxyglucose, suggesting that exocytosis of nanoparticles is an energy-dependent process. The nanoparticle retention increased with increasing nanoparticle dose in the medium but the effect was relatively less significant with the increase in incubation time. Interestingly, the exocytosis of nanoparticles was almost completely inhibited when the medium was depleted of serum. Further studies suggest that the addition of BSA in the serum free medium with or without platelet derived growth factor (PDGF) induced exocytosis of nanoparticles. The above result suggests that the protein in the medium is either adsorbed onto nanoparticles and/or carried along with nanoparticles inside the cells, which probably interacts with the exocytic pathway and leads to greater exocytosis of nanoparticles.nConclusions. The study demonstrated that endocytosis and exocytosis of nanoparticles are dynamic and energy-dependent processes. Better understanding of the mechanisms of endocytosis and exocytosis, studies determining the effect of nanoparticle formulation and composition that may affect both the processes, and characterization of intracellular distribution of nanoparticles with surface modifications would be useful in exploring nanoparticles for intracellular delivery of therapeutic agents.

Fluorescence and electron microscopy probes for cellular and tissue uptake of poly(D, L-lactide-co-glycolide) nanoparticles

Nanoparticles formulated from poly(D,L-lactide-co-glycolide) (PLGA) and poly(lactide) (PLA) are being extensively investigated for different therapeutic applications such as for sustained drug, vaccine, and gene delivery. For many of these applications, it is necessary to study the intracellular distribution as well as the tissue uptake of nanoparticles to optimize the efficacy of the encapsulated therapeutic agent. Fluorescence and electron microscopic techniques are usually used for the above purposes. Colloidal gold particles and fluorescent polystyrene, which are generally used as model particles for electron and fluorescence microscopy, respectively, may not be suitable alternatives to PLGA/PLA nanoparticles for these studies mainly because of the differences in their physical properties and also because they do not contain any therapeutic agent. The aim of the present study was to develop and characterize PLGA nanoparticle formulations that would be suitable for confocal/fluorescence and transmission electron microscopic (TEM) studies. Towards this objective, PLGA nanoparticles containing 6-coumarin as a fluorescent marker and osmium tetroxide as an electron microscopic marker with bovine serum albumin (BSA) as a model protein were formulated. Different physical properties of marker-loaded nanoparticles such as particle size, zeta potential, residual PVA content and protein-loading were compared with those of unloaded nanoparticles and were found to be not significantly different. Furthermore, marker-loaded nanoparticle formulations were non-toxic to the cells as unloaded nanoparticles. Nanoparticles loaded with 6-coumarin were found to be useful for studying intracellular nanoparticle uptake and distribution using confocal microscopy while osmium tetroxide-loaded nanoparticles were found to be useful for studying nanoparticle uptake and distribution in cells and tissue using TEM. It was concluded that 6-coumarin and osmium tetroxide could serve as useful fluorescence and electron microscopy probes, respectively, for incorporation into nanoparticles to study their cellular and tissue distribution.

The use of nanoparticle-mediated targeted gene silencing and drug delivery to overcome tumor drug resistance.

Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) enables cancer cells to develop resistance to multiple anticancer drugs. Functional inhibitors of P-gp have shown promising efficacy in early clinical trials, but their long-term safety is yet to be established. A novel approach to overcome drug resistance is to use siRNA-mediated RNA interference to silence the expression of the efflux transporter. Because P-gp plays an important role in the physiological regulation of endogenous and xenobiotic compounds in the body, it is important to deliver P-gp targeted siRNA and anticancer drug specifically to tumor cells. Further, for optimal synergy, both the drug and siRNA may need to be temporally colocalized in the tumor cells. In the current study, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel, along with P-gp targeted siRNA, using poly(D,L-lactide-co-glycolide) nanoparticles to overcome tumor drug resistance. Nanoparticles were surface functionalized with biotin for active tumor targeting. Dual agent nanoparticles encapsulating the combination of paclitaxel and P-gp targeted siRNA showed significantly higher cytotoxicity in vitro than nanoparticles loaded with paclitaxel alone. Enhanced therapeutic efficacy of dual agent nanoparticles could be correlated with effective silencing of the MDR1 gene that encodes for P-gp and with increased accumulation of paclitaxel in drug-resistant tumor cells. In vivo studies in a mouse model of drug-resistant tumor demonstrated significantly greater inhibition of tumor growth following treatment with biotin-functionalized nanoparticles encapsulating both paclitaxel and P-gp targeted siRNA at a paclitaxel dose that was ineffective in the absence of gene silencing. These results suggest that that the combination of P-gp gene silencing and cytotoxic drug delivery using targeted nanoparticles can overcome tumor drug resistance.

Nanoparticle-mediated simultaneous and targeted delivery of paclitaxel and tariquidar overcomes tumor drug resistance

Drug resistance is a major obstacle to the success of cancer chemotherapy. Overexpression of the drug-efflux transporter P-glycoprotein (P-gp) is a key factor contributing to tumor drug resistance. Third generation P-gp inhibitors like tariquidar have shown promising efficacy in early clinical trials. However, for maximum efficacy, it is important to limit the exposure of normal cells and tissues to the efflux inhibitor and the anticancer drug, and temporally colocalize the drug-inhibitor combination in the tumor cells. In this study, we investigated simultaneous and targeted delivery of anticancer drug, paclitaxel, with P-gp modulator, tariquidar, using poly(d,l-lactide-co-glycolide) nanoparticles to overcome tumor drug resistance. Nanoparticles were surface functionalized with biotin for active tumor targeting. Dual agent nanoparticles encapsulating the combination of paclitaxel and tariquidar showed significantly higher cytotoxicity in vitro than nanoparticles loaded with paclitaxel alone. Enhanced therapeutic efficacy of dual agent nanoparticles could be correlated with increased accumulation of paclitaxel in drug-resistant tumor cells. In vivo studies in a mouse model of drug-resistant tumor demonstrated significantly greater inhibition of tumor growth following treatment with biotin-functionalized nanoparticles encapsulating both paclitaxel and tariquidar at a paclitaxel dose that was ineffective in the absence of tariquidar. Taken together, these results suggest that the use of targeted, dual agent nanoparticles delivering a combination of P-gp modulator and anticancer drug is a very promising approach to overcome tumor drug resistance.

Polymeric nanoparticles for siRNA delivery and gene silencing

Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles formulated using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA) for siRNA delivery. A cationic polymer, polyethylenimine (PEI), was incorporated in the PLGA matrix to improve siRNA encapsulation in PLGA nanoparticles. PLGA-PEI nanoparticles were formulated using double emulsion-solvent evaporation technique and characterized for siRNA encapsulation and in vitro release. The effectiveness of siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire-fly luciferase, was investigated in cell culture. Presence of PEI in PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. PLGA-PEI nanoparticles carrying luciferase-targeted siRNA enabled effective silencing of the gene in cells stably expressing luciferase as well as in cells that could be induced to overexpress the gene. Quantitative studies indicated that presence of PEI in PLGA nanoparticles resulted in 2-fold higher cellular uptake of nanoparticles while fluorescence microscopy studies showed that PLGA-PEI nanoparticles delivered the encapsulated siRNA in the cellular cytoplasm; both higher uptake and greater cytosolic delivery could have contributed to the gene silencing effectiveness of PLGA-PEI nanoparticles. Serum stability and lack of cytotoxicity further add to the potential of PLGA-PEI nanoparticles in gene silencing-based therapeutic applications.