Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex.

Despite the importance of siRNA delivery systems, understanding of their intracellular fate remains elusive. We recently developed a multi-component siRNA nanocomplex to deliver siRNA to hepatic stellate cells (HSCs). The objective of this study is to study post-internalization trafficking of this siRNA nanocomplex and its multiple components like siRNA, protamine, and streptavidin, in HSCs. After internalization, the nanocomplex entrapped in early endosomes undergoes three possible routes including endosomal escape, exocytosis, and entrapment in lysosomes. Significant amount of siRNA dissociates from the nanocomplex to exert silencing activity. After escaping from endosomes, protamine dissociates from the nanocomplex and stays inside the cytoplasm. Golgi complex plays an important role in exocytosis of the nanocomplex. We also demonstrate that exocytosis is one of the major reasons accounting for the transient silencing activity of nonviral siRNA delivery. Incorporation of exocytosis inhibitors in nonviral siRNA delivery systems may extend the silencing activity of siRNA.

Peptides used in the delivery of small noncoding RNA.

RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed.

Development of streptavidin-based nanocomplex for siRNA delivery.

In our previous study, we have identified a PCBP2 siRNA that exhibits antifibrotic activity in rat hepatic stellate cells (HSCs) by inhibition of αCP2, a protein responsible for stabilization of the collagen α1 (I) mRNA in alcoholic liver fibrosis. This study aims to develop a streptavidin-based nanocomplex that can efficiently deliver the PCBP2 siRNA to HSCs. Biotin-siRNA and biotin-cholesterol were mixed with streptavidin to form the streptavidin-biotin complex, which was further condensed electrostatically with positively charged protamine to form the final multicomponent siRNA nanocomplex in the size range of 150-250 nm. The siRNA nanocomplex does not induce cytotoxicity in rat HSCs as compared to commercially available transfection agents. The cellular uptake efficiency of the siRNA nanocomplex is higher in rat HSCs than other cell lines, such as Caco-2 and PC-3, indicating that receptor-mediated endocytosis mainly contributes to the cellular uptake of the siRNA nanocomplex. The siRNA nanocomplex exhibits more than 85% silencing effect on the PCBP2 mRNA in HSCs. Stability study indicates that the nanocomplex can efficiently protect siRNA from degradation in the serum. The streptavidin-based multicomponent siRNA nanocomplex provides a promising strategy to deliver the PCBP2 siRNA to HSCs. Moreover, the nanocomplex can be used as a platform for other diseases by changing the siRNA sequence and targeting ligand.

Development of a peptide-drug conjugate for prostate cancer therapy.

TGX-221 is a highly potent phosphoinositide 3-kinase ß (PI3Kß) inhibitor that holds great promise as a novel chemotherapeutic agent to treat prostate cancer. However, poor solubility and lack of targetability limit its therapeutic applications. The objective of this present study is to develop a peptide-drug conjugate to specifically deliver TGX-221 to HER2 overexpressing prostate cancer cells. Four TGX-221 derivatives with added hydroxyl groups were synthesized for peptide conjugation. Among them, TGX-D1 exhibited a similar bioactivity to TGX-221, and it was selected for conjugation with a peptide promoiety containing a HER2-targeting ligand and a prostate specific antigen (PSA) substrate linkage. From this selection, the peptide-drug conjugate was proven to be gradually cleaved by PSA to release TGX-D1. Cellular uptake of the peptide-drug conjugate was significantly higher in prostate cancer cells compared to the parent drug. Moreover, both the peptide-drug conjugate and its cleaved products demonstrated comparable activities as the parent drug TGX-D1. Our results suggest that this peptide-drug conjugate may provide a promising chemotherapy for prostate cancer patients.

Identification of a LNCaP-specific binding peptide using phage display.

PURPOSE: To identify a LNCaP-specific peptide using a phage display library and evaluate its potential applications in targeted drug delivery. METHODS: Binding abilities of selected phages were evaluated by cell phage ELISA. The KYL peptide encoded by the most specific phage clone was synthesized, labeled with fluorescein, and assayed in various cell lines. A fusion peptide composed of the KYL peptide and a proapoptotic peptide ( D )(KLAKLAK)(2) was synthesized, and the cell death effect was evaluated on different cells. Moreover, the KYL peptide was conjugated to a cationic protein, protamine, to explore its potential application in siRNA delivery. RESULTS: One phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein-labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein-labeled siRNA into LNCaP cells. CONCLUSION: We identified a LNCaP-specific peptide and demonstrated its potential applications in targeted drug delivery to LNCaP cells.

PCBP2 siRNA reverses the alcohol-induced pro-fibrogenic effects in hepatic stellate cells.

PURPOSE: Type I collagen accumulates during liver fibrosis primarily because α-complex protein-2 (αCP(2)), encoded by the poly(rC) binding protein 2 (PCBP2) gene, binds to the 3' end of the collagen mRNA and increases its half-life. This study aimed to reverse the pro-fibrogenic effect of alcohol on hepatic stellate cells (HSCs) by silencing the PCBP2 gene with siRNA. METHODS: The silencing effects of a series of predesigned PCBP2 siRNAs were evaluated in the rat hepatic stellate cell line, HSC-T6. The pro-fibrogenic effects of alcohol on the expression levels of PCBP2 and type-I collagen were examined by several methods. The effect of PCBP2 siRNA on the stability of type I collagen α1(I) mRNA was investigated by an in vitro mRNA decay assay. RESULTS: We identified one potent PCBP2 siRNA that reversed the alcohol-induced expression of PCBP2 in HSCs. The decay rate of the collagen α1(I) mRNA increased significantly in HSCs treated with the PCBP2 siRNA. CONCLUSION: This study provides the first evidence that alcohol up-regulates the expression of PCBP2, which subsequently increases the half-life of collagen α1(I) mRNA. Silencing of PCBP2 using siRNA may provide a promising strategy to reverse the alcohol-induced pro-fibrogenic effects in HSCs.

Formulation development of a live attenuated human rotavirus (RV3-BB) vaccine candidate for use in low- and middle-income countries.

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2-8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50-60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitro gastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2-8°C and -20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities.

Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery.

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2-8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery.

Combined semi-empirical screening and design of experiments (DOE) approach to identify candidate formulations of a lyophilized live attenuated tetravalent viral vaccine candidate.

A combination experimental approach, utilizing semi-empirical excipient screening followed by statistical modeling using design of experiments (DOE), was undertaken to identify stabilizing candidate formulations for a lyophilized live attenuated Flavivirus vaccine candidate. Various potential pharmaceutical compounds used in either marketed or investigative live attenuated viral vaccine formulations were first identified. The ability of additives from different categories of excipients, either alone or in combination, were then evaluated for their ability to stabilize virus against freeze-thaw, freeze-drying, and accelerated storage (25°C) stresses by measuring infectious virus titer. An exploratory data analysis and predictive DOE modeling approach was subsequently undertaken to gain a better understanding of the interplay between the key excipients and stability of virus as well as to determine which combinations were interacting to improve virus stability. The lead excipient combinations were identified and tested for stabilizing effects using a tetravalent mixture of viruses in accelerated and real time (2-8°C) stability studies. This work demonstrates the utility of combining semi-empirical excipient screening and DOE experimental design strategies in the formulation development of lyophilized live attenuated viral vaccine candidates.