Skip to main content

Advertisement

SpringerLink
Log in

Enzyme Responsive Delivery of Anti-Retroviral Peptide via Smart Hydrogel

  • Research Article
  • Published:
AAPS PharmSciTech Aims and scope Submit manuscript

Abstract

In response to an urgent need for advanced formulations for the delivery of anti-retrovirals, a stimuli-sensitive hydrogel formulation that intravaginally delivers HIV-1 entry inhibitor upon being exposed to a specific protease was developed. The hydrogel formulation consists of PEG-azide and PEG-DBCO covalently linked to the entry inhibitor peptide, enfuvirtide, via substrate linker that is designed to undergo proteolysis by prostate specific antigen (PSA) present in seminal fluid and release innate enfuvirtide. Of the tested PSA substrate linkers (HSSKLQYY, GISSFYSSK, AYLMYY, and AYLMGRR), HSSKLQ was found to be an optimal candidate for PEG-based hydrogel with kcat/KM of 2.2 M−1 s−1. The PEG-based hydrogel displayed a pseudoplastic, thixotropic behavior with overall viscosity varying between 1516 and 2.2 Pa.s, within the biologically relevant shear rates of 0.01–100 s−1. It also exhibited viscoelastic properties appropriate for uniform spreading and being retained in vagina. PEG-based hydrogels were loaded with N3-HSSKLQ-enfuvirtide (HF42) that is customarily synthesized enfuvirtide prodrug with its N-terminus connected to HSSKLQ linker. The stimuli-sensitive PEG-based hydrogel formulations upon being exposed to PSA released 36.5 ± 4.8% of enfuvirtide over 24 h in human ejaculate mimic of vaginal simulant fluid and seminal simulant fluid mixed in 1:3 ratio, which is significantly greater than its IC50. The PEG-based hydrogel was non-cytotoxic to both vaginal epithelial cells (VK2/E6E7) and murine macrophages (RAW 264.7) and did not significantly induce the production of nitric oxide, an inflammatory mediator. The PEG-based hydrogel is found to have suitable physicochemical properties for an intravaginal formulation of the PSA substrate–linked anti-retrovirals and is safe towards vaginal epithelium. It is capable of delivering enfuvirtide with effective concentrations to prevent women from HIV-1 infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Data Availability

The authors confirm that the data supporting the findings of this study are available on request. Raw data were generated at UMKC. Derived data supporting the findings of this study are available from the corresponding author [C Lee] on request.

References

  1. Global HIV & AIDS statistics — 2020 fact sheet [Internet]. [cited 2021 Jan 3]. Available from: https://www.unaids.org/en/resources/fact-sheet. Accessed 16 Aug 2022

  2. 20.1 million girls and women living with HIV [Internet]. [cited 2021 Jan 3]. Available from: https://www.unaids.org/en/resources/infographics/girls-and-women-living-with-HIV. Accessed 16 Aug 2022

  3. AIDSinfo | UNAIDS [Internet]. [cited 2020 Apr 8]. Available from: https://aidsinfo.unaids.org/. Accessed 16 Aug 2022

  4. Abbas UL, Hood G, Wetzel AW, Mellors JW. Factors influencing the emergence and spread of HIV drug resistance arising from rollout of antiretroviral pre-exposure prophylaxis (PrEP). PLoS ONE. 2011;6(4): e18165.

    Article  CAS  Google Scholar 

  5. Lehman DA, Baeten JM, McCoy CO, Weis JF, Peterson D, Mbara G, et al. Risk of drug resistance among persons acquiring HIV within a randomized clinical trial of single-or dual-agent preexposure prophylaxis. J Infect Dis. 2015;211(8):1211–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Barreto-de-Souza V, Arakelyan A, Margolis L, Vanpouille C. HIV-1 Vaginal transmission: cell-free or cell-associated virus? Am J Reprod Immunol. 2014 Jun;71(6):589–99.

    Article  CAS  Google Scholar 

  7. Aaron E, Blum C, Seidman D, Hoyt MJ, Simone J, Sullivan M, et al. Optimizing delivery of HIV preexposure prophylaxis for women in the United States. AIDS Patient Care STDS. 2018;32(1):16–23.

    Article  Google Scholar 

  8. Baeten JM, Hendrix CW, Hillier SL. Topical microbicides in HIV prevention: state of the promise. Annu Rev Med. 2020;71:361–77.

    Article  CAS  Google Scholar 

  9. Musara P, Milford C, Shapley-Quinn MK, Weinrib R, Mutero P, Odoom E, et al. Preferences and acceptability of vaginal delivery forms for HIV prevention among women, male partners and key informants in South Africa and Zimbabwe: qualitative findings. AIDS Behavior. 2020;1–15.

  10. Fang W, Mphoyi BI, Motake DR, Liu J, Li W, Mei F, et al. Efficacy, adherence and side effects of PrEP for HIV-1 prevention. IJB. 2019 Aug 29;11(4):80.

    Article  CAS  Google Scholar 

  11. Nadendla K, Friedman SH. Light control of protein solubility through isoelectric point modulation. J Am Chem Soc. 2017 Dec 13;139(49):17861–9.

    Article  CAS  Google Scholar 

  12. Nadendla K, Sarode BR, Friedman SH. Hydrophobic tags for highly efficient light-activated protein release. Mol Pharmaceutics. 2019 Jul 1;16(7):2922–8.

    Article  CAS  Google Scholar 

  13. Nadendla K, Sarode B, Friedman SH. Chemical modification of proteins with photocleavable groups. In: Methods in enzymology. Elsevier; 2019. p. 113–28.

  14. Aimetti AA, Machen AJ, Anseth KS. Poly (ethylene glycol) hydrogels formed by thiol-ene photopolymerization for enzyme-responsive protein delivery. Biomaterials. 2009;30(30):6048–54.

    Article  CAS  Google Scholar 

  15. Azagarsamy MA, Anseth KS. Wavelength-controlled photocleavage for the orthogonal and sequential release of multiple proteins. Angew Chem. 2013;125(51):14048–52.

    Article  Google Scholar 

  16. Wang TJ, Rittenhouse HG, Wolfert RL, Lynne CM, Brackett NL. PSA concentrations in seminal plasma. Clin Chem. 1998;44(4):895a–896.

    Article  Google Scholar 

  17. Chan W, White P. Fmoc solid phase peptide synthesis: a practical approach. Oxford University Press; 1999. 371 p.

  18. Nadendla VSK. Second Generation Photoactivated Insulins [Internet] [Ph.D.]. ProQuest dissertations and theses. [Ann Arbor]: University of Missouri - Kansas City; 2020. Available from: http://proxy.library.umkc.edu/login?url=https://www.proquest.com/dissertations-theses/second-generation-photoactivated-insulins/docview/2423778183/se-2?accountid=14589. Accessed 16 Aug 2022

  19. Hakala TA, Yates EV, Challa PK, Toprakcioglu Z, Nadendla K, Matak-Vinkovic D, et al. Accelerating reaction rates of biomolecules by using shear stress in artificial capillary systems. J Am Chem Soc. 2021 Oct 13;143(40):16401–10.

    Article  CAS  Google Scholar 

  20. Yeruva TK. Smart hydrogel for enzyme responsive vaginal delivery of anti-HIV peptide therapeutics [Internet] [Ph.D.]. ProQuest Dissertations and Theses. [Ann Arbor]: University of Missouri - Kansas City; 2021. Available from: https://www.proquest.com/dissertations-theses/smart-hydrogel-enzyme-responsive-vaginal-delivery/docview/2615117404/se-2?accountid=14696. Accessed 16 Aug 2022

  21. Denmeade SR, Lou W, Lövgren J, Malm J, Lilja H, Isaacs JT. Specific and efficient peptide substrates for assaying the proteolytic activity of prostate-specific antigen. Can Res. 1997;57(21):4924–30.

    CAS  Google Scholar 

  22. Yu T, Malcolm K, Woolfson D, Jones DS, Andrews GP. Vaginal gel drug delivery systems: understanding rheological characteristics and performance. Expert Opin Drug Deliv. 2011;8(10):1309–22.

    Article  CAS  Google Scholar 

  23. Zhang C, Zhang T, Oyler NA, Youan BBC. Direct and real-time quantification of tenofovir release from pH-sensitive microparticles into simulated biological fluids using 1H-NMR. J Pharm Sci. 2014 Apr;103(4):1170–7.

    Article  CAS  Google Scholar 

  24. Owen DH, Katz DF. A vaginal fluid simulant. Contraception. 1999 Feb 1;59(2):91–5.

    Article  CAS  Google Scholar 

  25. Patel IH, Zhang X, Nieforth K, Salgo M, Buss N. Pharmacokinetics, pharmacodynamics and drug interaction potential of enfuvirtide. Clin Pharmacokinet. 2005;44(2):175–86.

    Article  CAS  Google Scholar 

  26. Payne RW, Nayar R, Tarantino R, Terzo SD, Moschera J, Di J, et al. Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography. Peptide Science: Original Research on Biomolecules. 2006;84(5):527–33.

    Article  CAS  Google Scholar 

  27. Owen DH, Katz DF. A review of the physical and chemical properties of human semen and the formulation of a semen simulant. J Androl. 2005;26(4):459–69.

    Article  CAS  Google Scholar 

  28. Neves J, d, da Silva MV, Gonçalves MP, Amaral MH, Bahia MF. Rheological properties of vaginal hydrophilic polymer gels. Current Drug Delivery. 2009;6(1):83–92.

    Article  Google Scholar 

  29. Hochmeister M. Evaluation of Prostate-Specific Antigen (PSA) Membrane tests for the forensic identification of semen. 1997;

  30. Haase AT. Targeting early infection to prevent HIV-1 mucosal transmission. Nature. 2010 Mar;464(7286):217–23.

    Article  CAS  Google Scholar 

  31. Veazey RS, Ketas TA, Klasse PJ, Davison DK, Singletary M, Green LC, et al. Tropism-independent protection of macaques against vaginal transmission of three SHIVs by the HIV-1 fusion inhibitor T-1249. Proc Natl Acad Sci U S A. 2008/07/22 ed. 2008 Jul 29;105(30):10531–6.

  32. Zhang T, Sturgis TF, Youan BBC. pH-responsive nanoparticles releasing tenofovir intended for the prevention of HIV transmission. Eur J Pharm Biopharm. 2011;79(3):526–36.

    Article  CAS  Google Scholar 

  33. Coulibaly FS, Ezoulin MJ, Purohit SS, Ayon NJ, Oyler NA, Youan BBC. Layer-by-layer engineered microbicide drug delivery system targeting HIV-1 gp120: physicochemical and biological properties. Mol Pharm. 2017;14(10):3512–27.

    Article  CAS  Google Scholar 

  34. Agrahari V, Zhang C, Zhang T, Li W, Gounev TK, Oyler NA, et al. Hyaluronidase-sensitive nanoparticle templates for triggered release of HIV/AIDS microbicide in vitro. AAPS J. 2014;16(2):181–93.

    Article  CAS  Google Scholar 

  35. Mattsson JM, Ravela S, Hekim C, Jonsson M, Malm J, Närvänen A, et al. Proteolytic activity of prostate-specific antigen (PSA) towards protein substrates and effect of peptides stimulating PSA activity. PLoS ONE. 2014;9(9): e107819.

    Article  Google Scholar 

  36. Plant TM, Zeleznik AJ. (Eds.). Knobil and Neill’s physiology of reproduction (4th ed.). Academic Press; 2014;1:790–793.

  37. Clark MR, Aliyar HA, Lee C, won, Jay JI, Gupta KM, Watson KM, et al. Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles. Int J Pharm. 2011;413(1–2):10–8.

    Article  CAS  Google Scholar 

  38. LeBeau AM, Kostova M, Craik CS, Denmeade SR. Prostate-specific antigen: an overlooked candidate for the targeted treatment and selective imaging of prostate cancer. Biol Chem. 2010;391(4):333–43.

    Article  CAS  Google Scholar 

  39. Yang CF, Porter ES, Boths J, Kanyi D, Hsieh MC, Cooperman BS. Design of synthetic hexapeptide substrates for prostate-specific antigen using single-position minilibraries. J Pept Res. 1999;54(5):444–8.

    Article  CAS  Google Scholar 

  40. Debela M, Magdolen V, Schechter N, Valachova M, Lottspeich F, Craik CS, et al. Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences. J Biol Chem. 2006;281(35):25678–88.

    Article  CAS  Google Scholar 

  41. Nelson M, Arastéh K, Clotet B, Cooper DA, Henry K, Katlama C, et al. Durable efficacy of enfuvirtide over 48 weeks in heavily treatment-experienced HIV-1-infected patients in the T-20 versus optimized background regimen only 1 and 2 clinical trials. JAIDS Journal of Acquired Immune Deficiency Syndromes. 2005;40(4):404–12.

    Article  CAS  Google Scholar 

  42. Lazzarin A, Clotet B, Cooper D, Reynes J, Arastéh K, Nelson M, et al. Efficacy of enfuvirtide in patients infected with drug-resistant HIV-1 in Europe and Australia. N Engl J Med. 2003;348(22):2186–95.

    Article  CAS  Google Scholar 

  43. Lalezari JP, Henry K, O’Hearn M, Montaner JS, Piliero PJ, Trottier B, et al. Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N Engl J Med. 2003;348(22):2175–85.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Dr. Thomas Johnston for allowing us to use Shimadzu LC-2010A instrument. The authors acknowledge Drs. Zahra Niroobaksh and Reid Brenner for their assistance with rheological experiments and studies with FTIR spectrometer, respectively. The authors also acknowledge Dr. Donggao Zhao, the director of EM lab, UMKC school of Dentistry, for the electron microscopy service.

Funding

This study was supported by the research grant from the School of Graduate Studies in the University of Missouri-Kansas City, the Research bridge fund from Division of Pharmacology and Pharmaceutical Sciences, and the Graduate assistant fund from the UMKC women’s council.

Author information

Authors and Affiliations

  1. Division of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Missouri, 2464 Charlotte Street, HSB 4242, Kansas City, MO, 64108, USA

    Taj Yeruva & Chi H. Lee

Authors
  1. Taj Yeruva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chi H. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T. Yeruva: initiate, design the conception, and implementation of the work; also involved with the acquisition, analysis, and interpretation of data for the work.

C. Lee: design and drafting of the work, interpretation of data for the work, revising it critically for important intellectual content, and final approval of the version to be published.

Corresponding author

Correspondence to Chi H. Lee.

Ethics declarations

Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 495 KB)

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yeruva, T., Lee, C.H. Enzyme Responsive Delivery of Anti-Retroviral Peptide via Smart Hydrogel. AAPS PharmSciTech 23, 234 (2022). https://doi.org/10.1208/s12249-022-02391-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1208/s12249-022-02391-w

Keywords

Search

Navigation