Revolutionizing HIV Treatment: Long-Acting Injectable Supramolecular Polymeric Hydrogels


One of the most formidable challenges in the battle against HIV/AIDS is ensuring the consistent and long-term adherence to antiretroviral therapy (ART).

ART has revolutionized the management of HIV, allowing individuals living with the virus to lead healthier lives. However, the effectiveness of this treatment depends on the strict adherence to a daily pill regimen. Non-adherence can lead to viral rebound and the development of drug-resistant strains, jeopardizing the patient’s health and well-being.

To address this issue, researchers have been exploring novel approaches to develop long-acting injectable formulations that provide sustained drug release, eliminating the need for daily dosing and improving patient adherence. One breakthrough in this endeavor is the development of self-assembling drug amphiphiles (DAs) based on lamivudine, a water-soluble antiretroviral agent.

The Challenge of Long-Acting HIV Treatment

Traditional HIV treatment regimens often require daily pill consumption, which can be burdensome and challenging to adhere to consistently. For long-term therapeutic success, it is crucial that patients strictly adhere to their prescribed treatment schedules.

The consequences of non-adherence are dire, including the resurgence of viral replication, potential development of drug resistance, and an increased risk of progression to AIDS.

This challenge prompted the exploration of innovative strategies to develop long-acting injectable treatments that can maintain therapeutic drug levels in the body over extended periods.

Supramolecular Polymers: The Potential Solution

In the quest for long-acting HIV treatment, scientists have turned to supramolecular polymers – a class of molecules with unique self-assembling properties. These polymers can spontaneously form intricate three-dimensional networks under physiological conditions, making them ideal candidates for sustained drug delivery.

In the case of HIV treatment, this property is harnessed to design self-assembling drug amphiphiles (DAs) based on lamivudine, a well-established water-soluble antiretroviral agent and nucleoside reverse transcriptase inhibitor.

The Ingenious Design of ARV DAs

The designed antiretroviral (ARV) DAs are engineered with precision to leverage the remarkable properties of supramolecular polymers for long-acting drug delivery.

They contain three pairs of alternating hydrophobic valine (V) and hydrophilic lamivudine-modified lysine (K3TC) residues, with a variable number of glutamic acids (E) positioned on the C-terminus.

This molecular arrangement allows for the spontaneous self-assembly of the ARV DAs into supramolecular filaments when dissolved in deionized water, resulting in structures several micrometers in length with varying degrees of lateral stacking.

Triggering Controlled Release

A key aspect of this breakthrough is the ability to trigger controlled drug release from these supramolecular filaments. The addition of 1× PBS (phosphate-buffered saline) induces immediate gelation in the ARV DAs containing 2 or 3 E residues. This gelation process is pivotal for long-acting drug delivery, as it allows the ARV DAs to be injected as a liquid and then solidify in situ, forming a hydrogel. Upon dilution in an in vitro setting, these hydrogels disassemble from their supramolecular state into monomeric units, facilitating a controlled, linear release of the antiretroviral drug.

In Vivo Success

The application of this innovative approach goes beyond theory and laboratory experiments. In vivo studies have validated the effectiveness of these ARV DAs. These studies confirmed that the injectable ARV supramolecular polymeric hydrogel not only maintained an extended therapeutic release but also demonstrated several other promising features:

  • Injectability: The hydrogel was easily injectable, a crucial factor for practical use.
  • Rapid In Situ Hydrogel Formation: The hydrogel formed rapidly in the injection site, ensuring that the drug is delivered precisely where needed.
  • Enhanced Local Retention: The unique properties of the hydrogel enabled it to stay at the injection site, enhancing the drug’s concentration in the target area.
  • Long-Acting Therapeutic Release: Over the course of a month, the ARV supramolecular polymeric hydrogel maintained a plasma concentration of lamivudine above its IC50 value, demonstrating its long-acting capabilities.
  • Minimal Systemic Immunogenicity: Importantly, the hydrogel exhibited minimal systemic immunogenicity, reducing the risk of adverse reactions.

The Promise of ARV Supramolecular Hydrogels

The groundbreaking success of these ARV supramolecular polymeric hydrogels has significant implications for the field of HIV treatment. By addressing the challenge of patient adherence through long-acting injectables, this approach has the potential to improve the lives of countless individuals living with HIV.

It represents a novel, rational design strategy for developing long-acting injectables and may serve as a model for other therapies beyond HIV treatment.

As researchers continue to refine and expand upon this innovation, the future of HIV management looks increasingly promising.


The development of long-acting injectables for HIV treatment has long been a goal of the medical community, driven by the need to improve patient adherence and overall treatment efficacy. The utilization of supramolecular polymers and the creation of ARV supramolecular polymeric hydrogels based on lamivudine is a significant step forward in this endeavor.

The self-assembling drug amphiphiles (DAs) designed with precision have demonstrated their potential through controlled drug release, injectability, in situ hydrogel formation, local retention, and long-acting therapeutic release.

These innovations provide hope for improved HIV treatment outcomes, reducing the daily pill burden and increasing patient adherence. As research in this area continues, the promise of long-acting injectables using drug-based molecular assembly strategies offers the potential to revolutionize the landscape of HIV treatment.

Experimental Methodology and Techniques

The successful design and evaluation of self-assembling drug amphiphiles (DAs) based on lamivudine for long-acting HIV treatment requires a comprehensive understanding of the methods and techniques used in this study. This chapter provides a detailed account of the general considerations and experimental procedures involved in the synthesis, characterization, and evaluation of the ARV DAs.

Chemicals and Reagents

The foundation of this study lies in the quality of the chemicals and reagents used. To ensure the reliability and reproducibility of the results, great care was taken in selecting and sourcing the necessary materials. Rink Amide MBHA resin was procured from Chem-Impex in Wood Dale, IL. Fmoc-protected amino acids and HBTU, essential for the synthesis of the ARV DAs, were sourced from AAPPTec in Louisville, KY. Lamivudine (3TC) was obtained from Sigma-Aldrich. All other reagents were purchased from reputable suppliers such as Sigma-Aldrich, TCI, Alfa Aesar, and VWR and were used without further purification.

Analytical Techniques

  • Reverse-phase High-Performance Liquid Chromatography (HPLC): The separation and purification of synthesized compounds were carried out using a Varian PrepStar SD-1 HPLC system from Agilent Technologies. A Varian 440-LC collector was employed to collect purified fractions, which were subsequently lyophilized using a Labconco FreeZone 4.5L -50C Freeze Dryer based in Kansas City, MO.
  • Mass Spectrometry (MS): Mass spectrometric data were acquired using a Thermo Scientific LCQ Fleet ion-trap mass spectrometer located in Waltham, MA. MS analysis is crucial for confirming the identity and purity of synthesized compounds.
  • Nuclear Magnetic Resonance (NMR): Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer, allowing for the structural elucidation of compounds through the analysis of chemical shifts.

Molecular Self-Assembly and Transmission Electron Microscopy

The self-assembly of ARV DAs, a fundamental aspect of this study, is a critical process to ensure the formation of the desired supramolecular structures. This was achieved by dissolving the conjugates in de-ionized water at a concentration of 5 mM, followed by sonication for 5 minutes and aging for 24 hours at room temperature. For the preparation of samples for Transmission Electron Microscopy (TEM):

  • Conventional TEM: A 7.5 µL droplet of the stock solution was deposited onto a 400-mesh carbon-coated copper TEM grid, with excess solution wicked away using filter paper. Staining with a 2% uranyl acetate solution was performed, and the grid was allowed to dry before imaging. Conventional TEM images were obtained using a FEI Tecnai 12 TWIN TEM operating at 100 kV.
  • Cryogenic Transmission Electron Microscopy (Cryo-TEM): Cryo-TEM imaging was performed on 300-mesh lacey carbon-coated grids, pretreated to be hydrophilic. The vitrified samples were imaged using a FEI Eagle camera, with precautions taken to maintain a low temperature to prevent sublimation of vitreous water.

Circular Dichroism (CD)

To gain insights into the secondary structures and chiral properties of the ARV DAs, CD spectroscopy was employed. Stock solutions of ARV DAs were diluted to 100 µM, transferred to a 1 mm path length flat quartz cell, and their CD spectra recorded across the range of 200 to 300 nm using a Jasco J-710 spectropolarimeter. Solvent background signals were subtracted from each sample’s data, which was then normalized to DA concentration and path length.

Zeta Potential Measurement

Zeta potential measurements, performed using a Zetasizer Nano ZS90 from Malvern Instruments Ltd., UK, provide valuable information about the surface charge of the ARV DAs. Samples were diluted to 200 µM, loaded into capillary cells, and allowed to equilibrate before measurements. The results represent the average values from three replicate measurements, providing insights into changes in zeta potential over time.

Critical Micellization Concentration (CMC) Measurement

The determination of the critical micellization concentration (CMC) for ARV DAs is vital for understanding their self-assembly behavior. This measurement employed Nile Red as a hydrophobic probe. By recording emission spectra in a spectrophotometer at specific wavelengths, the CMC value was calculated based on the ratio of emission intensity at different wavelengths.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR was employed to study the molecular interactions and confirm the structural features of ARV DAs. A 10 mM ARV DA solution in D2O was prepared, and the spectroscopic analysis was conducted on a Thermo Nicolet iS10 FTIR Spectrometer, enabling the characterization of molecular functional groups.

Thioflavin T (ThT) Assay

The ThT assay was used to monitor the formation of amyloid-like structures in the ARV DAs. This assay, performed in 96-well plates, employed ThT fluorescence measurements to detect changes in structural properties as a function of concentration.

In the subsequent chapters of this study, the results and insights gained from these experimental techniques will be discussed in detail, shedding light on the rational design and potential of ARV supramolecular polymeric hydrogels for long-acting HIV treatment.

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