Biodegradable nanoparticles for cytosolic delivery of therapeutics

Abstract

Many therapeutics require efficient cytosolic delivery either because the receptors for those drugs are located in the cytosol or their site of action is an intracellular organelle that requires transport through the cytosolic compartment. To achieve efficient cytosolic delivery of therapeutics, different nanomaterials have been developed that consider the diverse physicochemical nature of therapeutics (macromolecule to small molecule; water soluble to water insoluble) and various membrane associated and intracellular barriers that these systems need to overcome to efficiently deliver and retain therapeutics in the cytoplasmic compartment. Our interest is in investigating PLGA and PLA-based nanoparticles for intracellular delivery of drugs and genes. The present review discusses the various aspects of our studies and emphasizes the need for understanding of the molecular mechanisms of intracellular trafficking of nanoparticles in order to develop an efficient cytosolic delivery system.

Figure 1. Schematic drawing of steps involved in cytosolic delivery of therapeutics using polymeric nanoparticles (NPs)

(1) Cellular association of NPs, (2) Internalization of NPs into the cells by endocytosis, (3) Endosomal escape of NPs, (4) Release of therapeutic in cytoplasm, (5) Cytosolic transport of therapeutic agent, (6) Degradation of drug either in lysosomes or in cytoplasm, (7) Exocytosis of NPs. Major barriers include: (A) Cellular uptake of NPs, (B) Endosomal escape of NPs, (C) Cytoplasmic transport of therapeutic/NPs, (D) Sustained therapeutic benefit. [PE: Primary endosomes, RE: Recycling endosomes, Endo-lys: Endo-lysosomes, Lys: Lysosomes, Solid circles represent polymeric NPs].

Figure 2. Prolonged retention of NPs in the cytoplasm of cells

Transmission electron microscopic pictures demonstrating the presence of NPs in the VSMCs on day 1 (A), 3 (B), 10 (C), and 14 (D) post-incubation. The bar is 500 nm long. NPs are indicated black spherical structures in the cytosol. These NPs were loaded with osmium tetraoxide for the purpose of contrast. Reprinted with permission from ref [45]. Copyright (2004) American Chemical Society.

Figure 3. Sustained cytoplasmic delivery of drugs with NPs

(a) Intracellular dexamethasone levels following treatment with tritiated dexamethasone in solution or in nanoparticles (formulations A and B). Data are means ± the standard error of the mean (n=3). The two formulations of NPs differed only in their ability to release different amounts of dexamethasone. (b) Inhibition of VSMC proliferation with dexamethasone in solution and encapsulated in NP formulations. Cell proliferation was measured using a MTS assay (CellTiter 96® AQueous, Promega, Madison, WI). MTS is chemically reduced by cells into formazan, which is soluble in tissue culture medium. The measurement of the absorbance of the formazan was carried out using 96 well microplates at 492nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells. Data are means ± the standard deviation (n= 6). Reprinted with permission from ref [45]. Copyright (2004) American Chemical Society.

Figure 4

a) Intracellular DNA delivery. DNA was labeled with TOTO (red) and nanoparticles contained a fluorescent dye (6-coumarin, green). Single-dose of DNA (either alone or in nanoparticles) was added to cells, the medium was changed at 2 days and then on every alternate day thereafter. Cells were observed under confocal microscope. Red color is due to DNA that is released from nanoparticles, green color is due to nanoparticles, and yellow color is due to co-localization of released DNA (red) and nanoparticles (green). b) RT-PCR data of cells transfected with wt-p53 gene: Lane 1: Molecular weight marker, Lane 2: p53 DNA-loaded NPs, Lane 3: p53(-ve) DNA loaded NPs, Lane 4: p53 DNA only; Lane 5: β-actin for p53 DNA loaded nanoparticles, Lane 6: β-actin for p53(-ve) DNA loaded nanoparticles, Lane 7: β-actin for p53 DNA. c) Antiproliferative activity of wt-p53 DNA: MDA-M435S cells were treated either with A: wt-p53-plasmid DNA or wt-p53 DNA-loaded nanoparticles (NP) or B: DNA-Lipofectamine™ complex. Reprinted with permission from ref. [46]. Copyright (2004) American Chemical Society.

Figure 5. Enhanced cellular uptake and intracellular retention of drug with Tf-conjugated NPs

(A) Uptake of Tf-conjugated NPs (NPs-Tf) and unconjugated NPs (NPs) in MCF-7 cells. To determine the competitive inhibition of uptake of Tf-conjugated NPs, an excess of free Tf was added to the medium prior to incubating cells with Tf-conjugated NPs. Data as mean±SEM (n=6), (*) p< 0.05 NPs-Tf + free Tf versus NPs. (**) p< 0.005 NPs-Tf versus NPs. (B) Exocytosis of Tf-conjugated and unconjugated NPs in MCF-7 cells. Cells were incubated with Tf conjugated NPs (gray) and unconjugated NPs (black) at 100 μg/mL concentration for 1 h, cells were washed, and then cells were incubated with fresh medium. This NP level was taken as the cellular uptake (0 h time point). In other wells, the cells were washed and incubated with medium, and were processed as above at different time points to determine intracellular retention of NPs. Reprinted with permission from ref [63]. Copyright (2004) American Chemical Society.

Figure 6. In vivo efficacy of Tf-conjugated paclitaxel-loaded NPs

Antitumor activity of Tx-NPs-Tf in a murine prostate tumor model. PC3 cells (2 × 10⁶ cells) were implanted s.c. in athymic nude mice. Tumor nodules were allowed to grow to diameter of about 50 mm³ prior to receiving different formulations as a single-dose treatment. Tx-NPs-Tf (•, 24 mg/kg; □, 12 mg/kg), Tx-NPs (×, 24 mg/kg), Tx-Cremophor® EL formulation (▲, 24 mg/kg), (◆) control NPs and (■) Cremophor® EL formulation. Data are means ± s.e.m., n=6. *p <0.005 Tx-NPs-Tf versus Tx-NPs and Tx-Cremophor® EL groups. Reprinted with permission from ref [64], Copyright (2004) Wiley-Liss, Inc., A Wiley Company.