Sanjeeb K. Sahoo

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)

Nanotech approaches to drug delivery and imaging

Nanotechnology, a multidisciplinary scientific undertaking, involves creation and utilization of materials, devices or systems on the nanometer scale. The field of nanotechnology is currently undergoing explosive development on many fronts. The technology is expected to create innovations and play a critical role in various biomedical applications, not only in drug delivery, but also in molecular imaging, biomarkers and biosensors. Target-specific drug therapy and methods for early diagnosis of pathologies are the priority research areas where nanotechnology would play a vital role. This review considers different nanotechnology-based drug delivery and imaging approaches, and their economic impact on pharmaceutical and biomedical industries.

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.

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.

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.

EFFICACY OF TRANSFERRIN-CONJUGATED PACLITAXEL-LOADED NANOPARTICLES IN A MURINE MODEL OF PROSTATE CANCER

Chemotherapy remains the preferred choice of treatment for prostate cancer but modest drug response and significant toxicity by conventional methods of administration limit their efficacy. In our study, we determined the efficacy of paclitaxel (Tx)‐loaded biodegradable nanoparticles (NPs) on tumor inhibition. We hypothesized that NPs following conjugation to transferrin (Tf) ligand (NPs‐Tf) would enhance the therapeutic efficacy of the encapsulated drug. The antiproliferative activity of NPs was determined in human prostate cancer cell line (PC3) and their effect on tumor inhibition in a murine model of prostate cancer. NPs (∼ 220 nm in diameter, 5.4% w/w drug loading) under in vitro conditions exhibited sustained release of the encapsulated drug (60% release in 60 days). The IC50 (concentration of drug for 50% inhibition of cell growth) of the drug with Tf‐conjugated NPs (Tx‐NPs‐Tf) was about 5‐fold lower than that with unconjugated NPs (Tx‐NPs) or drug in solution. Animals that received a single‐dose intratumoral injection of Tx‐NPs‐Tf (Tx dose = 24 mg/kg) demonstrated complete tumor regression and greater survival rate than those that received either Tx‐NPs or Tx‐Cremophor® EL formulation. In conclusion, sustained release NPs demonstrated greater antitumor activity following their conjugation to Tf ligand.

Nano-Sized Carriers for Drug Delivery

Drug delivery is an important issue, especially with a new generation of therapeutics, which are either unstable in the biological environment, have poor transport properties across biological membranes, are insoluble in water, or have very low bioavailability. Nano-sized drug carriers can address some of the above issues and enhance their therapeutic efficacy. Different types of nano-sized carriers, such as nanoparticles, nanowires, nanocages, dendrimers, etc., are being developed for various drug-delivery applications. The challenge is to determine the therapeutic dose of the drug formulated in a system, which could be significantly different from that of the drug nanocarrier. In this regard, a better understanding of the pathophysiology of the disease condition under consideration is critical so that one can select and design an appropriate drug carrier system that would deliver a therapeutic dose of the drug in the target tissue or a body compartment at a rate and for the duration that is therapeutically effective and can cure the disease.