Hepatocyte-targeted delivery of imiquimod reduces hepatitis B virus surface antigen.

Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma. Interferon-α (IFN-α) is included in the standard treatment for chronic hepatitis B (CHB). However, this therapy causes serious side effects. Delivering IFN-α selectively to the liver may enhance its efficacy and safety. Imiquimod (IMQ), a Toll-Like Receptor (TLR) 7 agonist, stimulates the release of IFN-α that exhibits potent antiviral activity. However, the poor solubility and tissue selectivity of IMQ limits its clinical use. Here, we demonstrated the use of lipid-based nanoparticles (LNPs) to deliver IMQ and increase the production of IFN-α in the liver. We encapsulated IMQ in two liver-targeted LNP formulations: phospholipid-free small unilamellar vesicles (PFSUVs) and DSPG-liposomes targeting the hepatocytes and the Kupffer cells, respectively. In vitro drug release/retention, in vivo pharmacokinetics, intrahepatic distribution, IFN-α production, and suppression of serum HBV surface antigen (HBsAg) were evaluated and compared for these two formulations. PFSUVs provided >95% encapsulation efficiency for IMQ at a drug-to-lipid ratio (D/L) of 1/20 (w/w) and displayed stable drug retention in the presence of serum. DSPG-IMQ showed 79% encapsulation of IMQ at 1/20 (D/L) and exhibited ∼30% burst release when incubated with serum. Within the liver, PFSUVs showed high selectivity for the hepatocytes while DSPG-liposomes targeted the Kupffer cells. Finally, in an experimental HBV mouse model, PFSUVs significantly reduced serum levels of HBsAg by 12-, 6.3- and 2.2-fold compared to the control, IFN-α, and DSPG-IMQ groups, respectively. The results suggest that the hepatocyte-targeted PFSUVs loaded with IMQ exhibit significant potential for enhancing therapy of CHB.

Chemically engineering the drug release rate of a PEG-paclitaxel conjugate using click and steric hindrance chemistries for optimal efficacy.

A small molecule drug with poor aqueous solubility can be conjugated to a hydrophilic polymer like poly(ethylene glycol) (PEG) to form an amphiphilic polymer-drug conjugate that self-assembles to form nanoparticles (NPs) with improved solubility and enhanced efficacy. This strategy has been extensively applied to improve the delivery of several small molecule drugs. However, very few reports have succeeded to tune the rate of drug release from these NPs. To the best of our knowledge, there have been no reports of utilizing click and steric hindrance chemistry to modulate the drug release of self-assembling polymer-drug conjugates. In this study, we utilized click chemistry to conjugate methoxy-PEG (mPEG) to an anti-tumor drug, paclitaxel (PTX). A focused library of PTX-Rx-mPEG (x = 0, 1, 2) conjugates were synthesized with different chemical modalities next to the cleavable ester bond to study the effect of increasing steric hindrance on the self-assembly process and the physicochemical properties of the resulting PTX-NPs. PTX-R0-mPEG had no added steric hindrance (x = 0; minimal), PTX-R1-mPEG consisted of two methyl groups (x = 1: moderate), and PTX-R2-mPEG consisted of a phenyl group (x = 2: significant). Drug release studies showed that PTX-NPs released PTX at a decreased rate with increasing steric hindrance. Pharmacokinetic studies showed that the AUC of released PTX from the moderate-release PTX-R1-NP was approximately 20-, 6-, and 3-fold higher than that from free PTX, PTX-R0-NP and PTX-R2-NP, respectively. As a result, among these different PTX formulations, PTX-R1-NP showed superior efficacy in inducing tumor regression and prolonging the animal survival. The tumors treated with PTX-R1-NP displayed the lowest tumor progression markers (Ki68 and CD31) and the highest apoptotic marker (TUNEL) compared to the others. This work emphasizes the importance of taking a systematic approach in designing self-assembling polymer drug conjugates and highlights the potential of utilizing steric hindrance as a tool to tune the drug release rate from such systems.

Interplay between the linker and polymer molecular weight of a self-assembling prodrug on the pharmacokinetics and therapeutic efficacy.

Poorly water-soluble small hydrophobic compounds can be conjugated to a hydrophilic polymer such as methoxypolyethylene glycol (mPEG) to form amphiphilic prodrugs that can self-assemble into nanoparticles (NPs) with increased aqueous solubility, prolonged circulation, and improved delivery. There have been numerous reports utilizing this strategy to improve delivery of small molecule drugs, but few reports take systematic, structure-activity relationship (SAR)-based approaches to develop optimal prodrug conjugates. Additionally, it is important to study interplay of different components within the conjugate, such as polymer molecular weight (M.W.) and linker to obtain optimal efficacy and safety. In this study, we developed a click chemistry platform to conjugate mPEG of three different M.W. (low: 550 Da; medium: 2000 Da; high: 5000 Da) to a small molecular anti-tumor drug, gambogic acid (GA) via two different linkers (ester: fast release; amide: slow release) to generate six distinct conjugates. NPs formed from conjugates of mPEG550 displayed significantly higher hemolytic toxicity compared to those with higher M.W. (<10%), regardless of the linker type. Drug release studies showed that NPs with an amide linker displayed insignificant drug release (<0.5% per day) compared to those with an ester linker (1-2% per day). NPs formed with mPEG5000 using an ester linker (5000-E-NP) possessed the optimal balance between prolonged circulation (223-fold higher AUC1-24 h than free GA) and sufficient drug release (1.68 ± 0.13% per day), leading to superior anti-tumor efficacy compared to other formulations, while the corresponding amides (5000-A-NP) displayed the most prolonged circulation but only moderate efficacy likely due to insufficient drug release. Our work highlights the importance of diligently studying SAR on drug conjugates to improve drug delivery and confirms the robustness of using the click platform to generate a conjugate library with chemical diversity.