Daniel W. Pack

Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres--a review.

PLGA microspheres are widely studied for controlled release drug delivery applications, and many models have been proposed to describe PLGA degradation and erosion and drug release from the bulk polymer. Autocatalysis is known to have a complex role in the dynamics of PLGA erosion and drug transport and can lead to size-dependent heterogeneities in otherwise uniformly bulk-eroding polymer microspheres. The aim of this review is to highlight mechanistic, mathematical models for drug release from PLGA microspheres that specifically address interactions between phenomena generally attributed to autocatalytic hydrolysis and mass transfer limitation effects. Predictions of drug release profiles by mechanistic models are useful for understanding mechanisms and designing drug release particles.

The effect of glycosaminoglycan content on polyethylenimine-based gene delivery within three-dimensional collagen-GAG scaffolds.

The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.

Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways

PurposeNon-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing.MethodsWe formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways.ResultsPEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms.ConclusionsThe well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.

Efficient in vitro gene delivery by hybrid biopolymer/virus nanobiovectors

Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9μg chitosan/10(9) M-VLPs at 10×10(9)M-VLPs/ml to 40μg chitosan/10(9) M-VLPs at 2.5×10(9)M-VLPs/ml, χ/M-VLPs were ~300-350nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms.

Intracellular trafficking of hybrid gene delivery vectors

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.

Derivation of an Analytical Solution to a Reaction-Diffusion Model for Autocatalytic Degradation and Erosion in Polymer Microspheres

A mathematical reaction-diffusion model is defined to describe the gradual decomposition of polymer microspheres composed of poly(D,L-lactic-co-glycolic acid) (PLGA) that are used for pharmaceutical drug delivery over extended periods of time. The partial differential equation (PDE) model treats simultaneous first-order generation due to chemical reaction and diffusion of reaction products in spherical geometry to capture the microsphere-size-dependent effects of autocatalysis on PLGA erosion that occurs when the microspheres are exposed to aqueous media such as biological fluids. The model is solved analytically for the concentration of the autocatalytic carboxylic acid end groups of the polymer chains that comprise the microspheres as a function of radial position and time. The analytical solution for the reaction and transport of the autocatalytic chemical species is useful for predicting the conditions under which drug release from PLGA microspheres transitions from diffusion-controlled to erosion-controlled release, for understanding the dynamic coupling between the PLGA degradation and erosion mechanisms, and for designing drug release particles. The model is the first to provide an analytical prediction for the dynamics and spatial heterogeneities of PLGA degradation and erosion within a spherical particle. The analytical solution is applicable to other spherical systems with simultaneous diffusive transport and first-order generation by reaction.

Pulsatile Protein Release from Monodisperse Liquid-Core Microcapsules of Controllable Shell Thickness

PurposePulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules.MethodsUsing precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19xa0μm.ResultsWhen using high molecular weight PLG (Mw 88xa0kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55–65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80–90% of the encapsulated BSA within 7xa0days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35xa0days.ConclusionsBiodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.

Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells

PurposesiRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency.MethodsLipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers.ResultsInhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process.ConclusionsMP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.

Rapid and facile quantitation of polyplex endocytic trafficking

ABSTRACT Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3′‐diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30 min post‐transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.

Prospects of siRNA applications in regenerative medicine

Small interfering RNA (siRNA) has established its reputation in the field of tissue engineering owing to its ability to silence the proteins that inhibit tissue regeneration. siRNA is capable of regulating cellular behavior during tissue regeneration processes. The concept of using siRNA technology in regenerative medicine derived from its ability to inhibit the expression of target genes involved in defective tissues and the possibility to induce the expression of tissue-inductive factors that improve the tissue regeneration process. To date, siRNA has been used as a suppressive biomolecule in different tissues, such as nervous tissue, bone, cartilage, heart, kidney, and liver. Moreover, various delivery systems have been applied in order to deliver siRNA to the target tissues. This review will provide an in-depth discussion on the development of siRNA and their delivery systems and mechanisms of action in different tissues.