Design and evaluation of a CXCR4 targeting peptide 4DV3 as an HIV entry inhibitor and a ligand for targeted drug delivery.

The feasibility of utilizing the cell surface chemokine receptor CXCR4 for human immunodeficiency virus (HIV) entry inhibition and as an intracellular portal for targeted drug delivery was evaluated. Novel DV3 ligands (1DV3, 2DV3, and 4DV3) were designed, synthesized and conjugated to various probes (fluorescein isothiocyanate (FITC) or biotin) and cargos with sizes ranging from 10 to 50 nm (polyethylene glycol (PEG), streptavidin, and a polymeric nanoparticle). 4DV3 conjugated probes inhibited HIV-1 entry into the CXCR4-expressing reporter cell line TZM-bl (IC50 at 553 nM) whereas 1DV3 and 2DV3 did not. 4DV3 also inhibited binding of anti-CXCR4 antibody 44,708 to TZM-bl cells with nanomolar potency, while the small-molecule CXCR4 antagonist AMD3100 did not. Molecular modeling suggested simultaneous binding of a single 4DV3 molecule to four CXCR4 molecules. Differences in CXCR4-binding sites could explain the discrete inhibitory effects observed for 4DV3, the 44,708 antibody and AMD3100. In the Sup-T1 cell chemotaxis assay, the 4DV3 ligand functioned as a CXCR4 allosteric enhancer. In addition, 4DV3 ligand-conjugated cargos with sizes ranging from 10 to 50 nm were taken up into CXCR4-expressing Sup-T1 and TZM-bl cells, demonstrating that CXCR4 could serve as a drug delivery portal for nanocarriers. The uptake of 4DV3 functionalized nanocarriers combined with the allosteric interaction with CXCR4 suggests enhanced endocytosis occurs when 4DV3 is the targeting ligand. The current results indicate that 4DV3 might serve as a prototype for a new type of dual function ligand, one that acts as a HIV-1 entry inhibitor and a CXCR4 drug delivery targeting ligand.

Colorectal delivery and retention of PEG-Amprenavir-Bac7 nanoconjugates--proof of concept for HIV mucosal pre-exposure prophylaxis.

Local delivery of anti-HIV drugs to the colorectal mucosa, a major site of HIV replication, and their retention within mucosal tissue would allow for a reduction in dose administered, reduced dosing frequency and minimal systemic exposure. The current report describes a mucosal pre-exposure prophylaxis (mPrEP) strategy that utilizes nanocarrier conjugates (NC) consisting of poly(ethylene glycol) (PEG), amprenavir (APV), and a cell-penetrating peptide (CPP; namely Bac7, a fragment derived from bactenecin 7). APV-PEG NCs with linear PEGs (2, 5, 10, and 30 kDa) exhibited reduced (52-21%) anti-HIV-1 protease (PR) activity as compared to free APV in an enzyme-based FRET assay. In MT-2 T cells, APV-PEG3.4 kDa-FITC (APF) anti-HIV-1 activity was significantly reduced (160-fold, IC50 = 8064 nM) due to poor cell uptake, whereas it was restored (IC50 = 78.29 nM) and similar to APV (IC50 = 50.29 nM) with the addition of Bac7 to the NC (i.e., APV-PEG3.4 kDa-Bac7, APB). Flow cytometry and confocal microscopy demonstrated Bac7-PEG3.4 kDa-FITC (BPF) uptake was two- and fourfold higher than APF in MT-2 T cells and Caco-2 intestinal epithelial cells, respectively. There was no detectable punctate fluorescence in either cell line suggesting that BPF directly enters the cytosol thus avoiding endosomal entrapment. After colorectal administration in mice, BPF mucosal concentrations were 21-fold higher than APF concentrations. BPF concentrations also remained constant for the 5 days of the study suggesting that (1) the NC's structural characteristics (i.e., the size of the PEG carrier and the presence of a CPP) significantly influenced tissue persistence, and (2) the NCs were probably lodged in the lamina propria since the average rodent colon mucosal cell turnover time is 2-3 days. These encouraging results suggest that Bac7 functionalized NCs delivered locally to the colorectal mucosa may form drug delivery depots that are capable of sustaining colorectal drug concentrations. Although the exact mechanisms for tissue persistence are unclear and will require further study, these results provide proof-of-concept feasibility for mPrEP.

Multimeric peptide-based PEG nanocarriers with programmable elimination properties.

In the current study, the design, synthetic feasibility and biochemical characterization of biodegradable peptidic PEG-based nanocarriers are described. The components were selected to influence the body elimination pathway upon nanocarrier biodegradation. Two prototypical nanocarriers were prepared using non-PEGylated and PEGylated peptidic cores [CH(3)CO-(Lys-betaAla-betaAla)(X)-Cys-CONH(2) (X=2, 4)]. A homodimeric nanocarrier with 4 copies of fluorescein-PEG5kDa was synthesized by linking two PEGylated peptidic cores (X=2) using a disulfide bond. A dual labeled heterodimeric nanocarrier with 2 copies of fluorescein-PEG5kDa and 4 copies of Texas Red was also synthesized. Optimum conditions for linking imaging agents, PEG, or a peptidic core to a peptidic core were determined. Significantly higher yields (69% versus 30%) of the PEGylated peptidic core were obtained by using 2 copies of beta-alanine as a spacer along with increasing DMSO concentrations, which resulted in reduced steric hindrance. Stoichiometric addition of the components was also demonstrated and found to be important for reducing polydispersity. Nanocarrier biodegradation was evaluated in simulated intracellular and extracellular/blood environments using 3 mm and 10 microm glutathione in buffer, respectively. The nanocarrier was 9-fold more stable in the extracellular environment. The results suggest selective intracellular degradation of the nanocarrier into components with known body elimination pathways.

Optimizing size and copy number for PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarrier uptake by macrophages.

Curing HIV-1 infection has remained elusive because of low and fluctuating drug levels arising from poor absorption, the development of viral reservoirs and sanctuary sites, toxicity, and patient nonadherence. The present study addresses the issue of insufficient drug exposure in macrophages. Viral reservoir sites such as macrophages are believed to be responsible for the viral rebound effect observed upon the discontinuation of anti-HIV drug therapy. In our proposed model, a drug can be covalently attached to a nanocarrier in order to facilitate the delivery of therapeutic agents to the site(s) of infection. As an initial step, we propose the covalent attachment of several copies of N-formyl-Met-Leu-Phe (fMLF), a known chemo-attractant for macrophages. In this article, one or more copies of fMLF were conjugated to multifunctional commercially available or novel, peptide-based PEG nanocarriers in which the structure was varied by appending PEGs with average molecular weights of 5, 20, and 40 kDa. U937 cell-specific binding and cellular uptake were analyzed. The results of uptake studies indicate that (i) uptake is energy dependent and mediated by a fMLF receptor, (ii) appending only 2 copies of the targeting ligand to the multifunctional nanocarrier appears sufficient for binding in vitro, and (iii) of the three configurations studied, the nanocarrier with a molecular weight of about 20 kDa, corresponding to a size of 20-60 nm, demonstrated the highest uptake. The results of the current studies demonstrate the feasibility of targeting macrophages and the suitability of using these synthetically versatile peptide--backbone PEG nanocarriers. The convenience, flexibility and possible limitations of this nanocarrier approach are discussed.