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Co-delivery of HIV-1 entry inhibitor and NNRTI shuttled by nanoparticles: cocktail therapeutic strategy for antiviral therapy - "new strategies for HIV cure" - Nanotechnology & ARTs
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There are 2 articles here, the commentary immediately below followed by the main study......
Co-delivery of HIV-1 entry inhibitor and NNRTI shuttled by nanoparticles: cocktail therapeutic strategy for antiviral therapy....."In this study, a new cocktail-like nanoparticle drug delivery system was developed by conjugating an HIV-1 entry inhibitor, T1144 peptide"
NanoART ATV/r + URMC-099, a Neuroprotective agent, Additively Reduce HIV Activity....... - (01/27/16)
"new strategies for HIV cure" - Nanotechnology & ARTs
"the authors of the current article have already demonstrated that encapsulation of DAAN15h in PEG-polymeric nanoparticles increased its antiviral activities on CD4, CCR5 or CXCR4–positive cells, enhancing its pharmacokinetic profile [10]. Now they suggest for the first time the nanoformulation of the NNRTI 14f and the fusion inhibitor T1144. This association in one single nanodevice of anti-transcriptase activity and HIV fusion inhibition represents a real novelty. The multifunctional anti-HIV nano-cocktail will be able to inhibit HIV-1 fusion with cells through the activity of its surface T1144 peptide, which will enhance cellular uptake of 14f and thus provide sustained controlled release of the drug into the target cell. The beauty of this new concept of HIV therapy is evident and resides in the possibility of enhanced combination therapy which targets multiple key points of viral replication with greatly increased antiviral activity. From a clinical point of view, the strongly improved intracellular antiviral activity without cellular toxicity, the increased f14 blood concentration and circulation time upon in vivo administration, with promised efficacy on a wide spectrum of HIV-1 strains, including resistant ones is appealing.
Together, these nano-strategies could lead to the formulation of a completely new class of "intelligent" anti-HIV drugs, with the possibility of a combined antiretroviral approach for both extra- and intracellular reservoirs of HIV. Thus, nanotechnology offers a unique opportunity to combine and improve different pharmacological profiles of antiretroviral drugs, with more convenient drug administration and potentially better patient adherence to HIV therapy. For these reasons, we believe that this multivalent nano-platform, able to optimize drug delivery and pharmacokinetic properties while providing a combinatorial therapeutic effect on resistant HIV strains, represents a promising approach in the landscape of new strategies for HIV cure."
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"The emergence of nanotechnology has made a significant impact on the development of clinical therapeutics, particularly providing an unparalleled opportunity for development of anti-HIV drugs. The application of nanotechnology offers the superiority of using anti-HIV drugs at lower dose, reduced systemic toxicity and enhanced therapeutic efficacy. However, translation of nanotechnology-based anti-HIV therapy into the clinics remains challenging. Significant efforts are needed to promote the translation process, including: 1) selection of absolutely biocompatible and biodegradable composition of nanoparticles; 2) carefully and rationally tailoring size and physicochemical property of the nanoparticles; and 3) providing desirable control over the drug biodistribution and pharmacokinetics to improve efficacy and reduce toxicity of nanoparticle-based anti-HIV therapeutics.
In conclusion, we employed biodegradable organic nanoparticles,PEG-PLA-NP, for encapsulation of anti-HIV-1 drug 14f and conjugation of T1144, a third-generation HIV-1 entry inhibitor, on the surface of nanoparticles. Our results demonstrated that the combination of entry inhibitor with reverse transcriptase inhibitor was highly effective in inhibiting R5-HIV-1 and X4-HIV-1, primary HIV-1 isolates, NNRTI-resistant HIV-1 strains, and T1144-resistant HIV-1 strains. Additionally, T1144-NP-14f exhibited enhanced intracellular uptake, sustained controlled release behavior and prolonged blood circulation time. The results indicated that the new cocktail-like nanoparticle drug delivery platform could serve as an effective anti-HIV-1 regimen by taking advantage of extrinsic and intrinsic antiviral activity of the partner drugs."
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New perspectives on nanotechnology and antiretroviral drugs: a small solution for a big promise in HIV treatment?.
AIDS Jan 23 2016
Corsi, Fabio; Fiandra, Luisa; Rizzardini, Giuliano
In this issue Li W et al [1] present a well-designed multidisciplinary study, aimed to demonstrate the potential benefit of a multifunctional polymeric nanodevice for delivering HIV treatment. The study offers a new perspective on the use of nanotechnology for combination HIV management, since the proposed system is a smart biodegradable PLA-based nanoparticle with a double therapeutic function, encapsulates the non-nucleoside reverse transcriptase inhibitor 14f with the surface conjugated to the third-generation HIV-1 fusion inhibitor T1144.
Among antiretroviral drugs, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are considered valid therapeutic tools, even if their clinical application has been limited by some well-known factors such as the short half-life after administration and the rapid induction of drug resistance [2]. The less commonly used fusion inhibitors also are strongly limited by the unfavorable pharmacological profile of these peptides, but also by their high production costs, the difficult administration schedule and the induction of adverse effects [3]. Beyond the limitations of the single drugs, a combination of different antiviral activities is necessary for effective HIV therapy, which requires addressing multiple phases of HIV replication. For this reason, "cocktail" therapies are largely used in current clinical practice. Yet, the different pharmacological profiles, half-lives and bioavailability of the cocktail components may result in decrease global antiretroviral activity [3].
In this scenario, nanotechnology may play a relevant role. The potential advantages in using nanoparticles for treatment of HIV infection include their capability to incorporate, encapsulate, or conjugate a variety of known antiretrovirals, in order to optimize the pharmacological profiles of these drugs, their specific homing in infected cells, and their anti- HIV activity [2, 4]. In fact, the preclinical study with nanoformulated antiretroviral therapy has suggested that intracellular translocation of nanoparticles could significantly improve the anti- HIV activity of the drugs due to their compartmentalization in endosomes where HIV active replication occurs [5]. Moreover, nanoformulation has been demonstrated to allow drug penetration into HIV sanctuaries (e.g. central nervous system, genital tract, lymph nodes, latently infected CD4+ T cells, etc), for effective eradication of the virus, with a potentially substantial impact on HIV treatment and prevention [6]. Interestingly, it has been recently reported that even complex anti-HIV molecules can successfully penetrate into the blood brain barrier if conjugated to iron oxide nanoparticles coated with an amphiphilic polymer [7].
Concerning the optimization of reverse transcriptase inhibitor activity upon nanoformulation, recent studies have demonstrated the role of cationic nanogels-NRTIs targeted for the brain specific apolipoprotein E receptor in protection from neurotoxicity together with excellent antiviral activity in brain macrophages [8]. Also, regarding the issue of HIV sanctuaries, a recent review discussed the use of different types of nanoparticles to shuttle NRTIs and NNRTIs into HIV reservoirs, since the ability to deliver combination antiviral therapy into HIV sanctuaries will be probably the next major clinical challenge [9]. Moreover, the authors of the current article have already demonstrated that encapsulation of DAAN15h in PEG-polymeric nanoparticles increased its antiviral activities on CD4, CCR5 or CXCR4–positive cells, enhancing its pharmacokinetic profile [10]. Now they suggest for the first time the nanoformulation of the NNRTI 14f and the fusion inhibitor T1144. This association in one single nanodevice of anti-transcriptase activity and HIV fusion inhibition represents a real novelty. The multifunctional anti-HIV nano-cocktail will be able to inhibit HIV-1 fusion with cells through the activity of its surface T1144 peptide, which will enhance cellular uptake of 14f and thus provide sustained controlled release of the drug into the target cell. The beauty of this new concept of HIV therapy is evident and resides in the possibility of enhanced combination therapy which targets multiple key points of viral replication with greatly increased antiviral activity. From a clinical point of view, the strongly improved intracellular antiviral activity without cellular toxicity, the increased f14 blood concentration and circulation time upon in vivo administration, with promised efficacy on a wide spectrum of HIV-1 strains, including resistant ones is appealing.
Together, these nano-strategies could lead to the formulation of a completely new class of "intelligent" anti-HIV drugs, with the possibility of a combined antiretroviral approach for both extra- and intracellular reservoirs of HIV. Thus, nanotechnology offers a unique opportunity to combine and improve different pharmacological profiles of antiretroviral drugs, with more convenient drug administration and potentially better patient adherence to HIV therapy. For these reasons, we believe that this multivalent nano-platform, able to optimize drug delivery and pharmacokinetic properties while providing a combinatorial therapeutic effect on resistant HIV strains, represents a promising approach in the landscape of new strategies for HIV cure.
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Co-delivery of HIV-1 entry inhibitor and NNRTI shuttled by nanoparticles: cocktail therapeutic strategy for antiviral therapy
Abstract
Objectives: Traditionally, the antiviral efficacy of classic cocktail therapy is significantly limited by the distinct pharmacokinetic profiles of partner therapeutics that lead to inconsistent in vivo bio-distribution. Here we developed a new cocktail-like drug delivery vehicle using biodegradable polymeric nanoparticles (NP) encapsulating non-nucleoside reverse transcriptase inhibitor (NNRTI) DAAN-14f (14f), surface-conjugated with HIV-1 fusion inhibitor T1144, designated T1144-NP-DAAN-14f (T1144-NP-14f), and aiming to achieve enhanced cellular uptake, improved antiviral activity and prolonged blood circulation time.
Methods: T1144-NP-14f was prepared through the emulsion/solvent evaporation technique and a maleimide-thiol coupling reaction. Particle size and morphology were determined by dynamic light scattering detection and transmission electron microscopy. Anti-HIV-1 activity was assessed by HIV-1 Env-mediated cell-cell fusion and infection by laboratory-adapted, primary, and resistant HIV-1 isolates, respectively. The in vitro release of 14f was investigated using the equilibrium dialysis method, and the pharmacokinetic study of T1144-NP-14f was performed on Sprague-Dawley rats.
Results: T1144-NP-14f displayed a spherical shape under TEM observation and had a size of 117 +/- 19 nm. T1144-NP-14f exhibited strongest antiviral activity against a broad spectrum of HIV-1 strains, including NNRTI-, T1144-, or T20-resistant isolates, respectively. Both in vitro release and in vivo pharmacokinetic profile showed that T1144-NP-14f exhibited a sustained controlled release behavior.
Conclusions: Our results demonstrated that the combination of entry inhibitor with NNRTI encapsulated in nanoparticles (T1144-NP-14f) was highly effective in inhibiting HIV-1 infection. This new cocktail-like drug delivery platform could serve as an effective anti-HIV-1 regimen by taking advantage of the extrinsic and intrinsic antiviral activity of individual drugs.
Introduction
In recent years, human immunodeficiency virus type 1 (HIV-1) has become one of the deadliest infectious diseases among adults. Globally, acquired immune deficiency syndrome (AIDS) induced by HIV infection has killed more than 25 millionpeople (http://www.unaids.org).UNAIDS estimates about 33 million people living with HIV/AIDS worldwide, including approximately 4.5 million youths and 2.1 million adolescents. Although many infected individuals have access to highly active antiretroviral therapy (HAART), new drugs and drug delivery strategies that offer improvements in potency and activity against HIV, even against multidrug-resistant HIV-1 viruses, are urgently needed [1, 2].
To date, mostapproved HIV medicines belong tothe class ofreverse transcriptase inhibitorsand protease inhibitors [3].However, clinical application of these inhibitors has been largely limited by the short half-lifeafter administration, rapid induction of drugresistance, and high cost[4].For example, enfuvirtide (also known as T20), which was the first HIV fusion inhibitor approved by FDA, exhibited favorable anti-HIV efficacy by interfering with the target cell to block the fusion of viral and cellular membrane[1, 5].However, the drawbacks, including short in vivohalf-life(less than 3 h) and the emergence of T20-resistant strains, have significantly impededthe broadclinical application of T20 [6-9].Furthermore, the therapeutic potency of T20 cannot be increasingly enhanced by the lack of the gp41 pocket-binding domain.
Drugdelivery systems based on nanotechnologyhaveoffered approaches resulting in the development of a "diverse toolbox" fordisease treatment[10].A case in point is represented by therapeutic peptidesor small-moleculedrugs conjugated/incorporated into versatile nanoparticles that exhibit well-recognized advantages,including hydrophilic and hydrophobic multidrug co-incorporation, prolonged circulation time in vivo, decorationwith functionalized ligands,and enhanced patient compliance [11, 12].Among a myriadof successful applications, nanoparticles for anti-HIV treatment have emerged as an especially promising drug delivery platform in comparison to conventional drug delivery formulationsas a consequence ofdecreased drug clearance rate at physiological condition and controlled, site-specific drug release, thus reducing unwanted side effects [13, 14].More importantly, nanoparticle drug delivery strategies can effectively circumvent the P-glycoprotein (Pgp)efflux process, which can significantly enhance intracellular drug concentration [15]. Our previous study showed that the reverse transcriptase inhibitorDAAN-15h, which was encapsulated in PEG-PLA NP, exhibited high antiviral activities in MT-2, M7, and TZM-b1 cells expressing CD4 receptor and CCR5 or CXCR4 co-receptor compared to DAAN-15h drug solution [16]. Therefore, the integration of anti-HIV drugs with nanoparticles can broaden drug deliveryflexibility and the scope of drug design, allowing for promising applicationsintheanti-HIV treatment field.
Combination therapy holds considerable appeal in enhancingantiviral activity by achieving synergistic effectsand has beenvalidated asmore effectivethan monotherapy[17-20].However, the general administration ofa"cocktails"-based combination therapy often suffers from distinct pharmacokinetic profiles of different therapeutics that lead to inconsistent in vivobio-distribution and,hence,inefficient therapy[21-23].To address this dilemma, a new cocktail-like drug delivery vehicle using biodegradable nanoparticles was developed in this study. Specifically, wetook advantage of functional maleimide groups on PEG chains and employed biocompatible Mal-PEG-PLAto engineer the nanoparticles with encapsulated NNRTI-DAAN-14f (14f) andsurface-conjugated T1144 peptide, a third-generation HIV fusion inhibitorwith enhanced antiviral activity (Fig. 1a). As an extrinsic (i.e., extracellular) combinatorial component, peptide T1144 would be expected to bind to the exposed grooves on the pre-fusion intermediate (PFI) and inhibit the fusion of virus with the target cell membrane [24], whereas the intrinsic (i.e., intracellular) combinatorial component, encapsulated NNRTI 14f, would be expected to internalize, diffuse and interact with a highly hydrophobic cavity in reverse transcriptase in a noncompetitive manner and allosterically block thechemical step of DNA synthesis(Fig. 1b)[25, 26].As such, theintegration of T1144 and 14fin a single nanoparticle offers a combinatorial antiviral effect against a broad spectrum of HIV-1 strains, including multidrug-resistant mutants.Moreover, the new cocktail-like drug delivery vehicle T1144-NP-DAAN-14f (T1144-NP-14f) holds other advantages, including enhanced intracellular uptake, sustained controlled release behavior, and prolonged blood circulation time in vivo. We believe this new strategyfor anti-HIV treatment holdspromise in overcoming the limitations and drawbacks of conventional single anti-HIV drug delivery at both research and clinical levels.
Discussion
Nanotechnology has allowed versatile drug delivery platforms with high drug drug-loading capacity, as well as enhanced bioavailability and biocompatibility [39]. In this study, a new cocktail-like nanoparticle drug delivery system was developed by conjugating an HIV-1 entry inhibitor, T1144 peptide, on the surface of 14f-loaded NPs. This strategy addressed the current limitations for HIV-1 treatment, such as insufficient cellular internalization of drugs and short half-life in vivo. By combining the extracellular antiviral effect of T1144 peptide on the cell surface and the intracellular antiviral activity of 14f after internalization, we believe the new cocktail-like T1144-NP-14f drug delivery system could serve as an optimized therapeutic strategy to combat HIV-1 infection.
As shown in Figure 1, T1144 and 14f have different active destinations and, hence, the potential to block HIV-1 infection at different stages of the HIV-1 life cycle. T1144, as an entry inhibitor, could interact with gp41 NHRs and form 6-HB with N peptide (N36) to inhibit HIV-1 gp41-mediated virus-cell fusion, while the NNRTI-14f could interact with a highly hydrophobic cavity in reverse transcriptase (RT) in a noncompetitive mannerto blockthechemical step of DNA synthesis and, subsequently, block viral replication. Targeting multiple steps during the process of HIV-1 infection can reduce the development of new resistant viral strains [40, 41].
T1144-NP-14f nanoparticles were obtained through an emulsion/evaporation method with diameters of approximately 100 nm and an average narrow distribution [42, 43]. The cellular uptake and membrane transport mechanism of T1144-NP-14f showed significantly higher cellular internalization of T1144-NP-coumarin-6 and NP-coumarin-6 when compared with coumarin-6 solution (Fig.3), indicating that the NP carrier could enhance the endocytosis of drug and T1144 peptide, but without affecting interaction between functionalized nanoparticles and MT-2 or M7 cells. Successful intracellular drug delivery is a key step to improve the efficiency of a nanoparticle-base therapeutic. To figure out the endocytosis pathway, we incubated MT-2 or M7 cells with several different inhibitors to reveal the endocytosis mechanism of T1144-NP-coumarin-6. The internalization of both NP-coumarin-6 and T1144-NP-coumarin-6 was significantly inhibited by NaN3+deoxyglucose,M-β-CD, and monensin, suggesting that the endocytosis of both NP-coumarin-6 and T1144-NP-coumarin-6 was lipid raft-mediated and energy-dependent with lysosome-enabled transport [44-46]. Furthermore, confocal images showed the extensive distribution of NP after endocytosis at cytoplasm, which was the active destination of NNRTIs, as evidenced by the separated fluorescence of coumarin-6 and DAPI (Fig. S2, ). More importantly, the separated fluorescence of nanoparticle and lysosome indicated the efficient endosome escape, which was beneficial for intracellular functional drugs since the drugs entrapped in the endosome would be degraded. Taken together, the enhanced cellular uptake and efficient endosome escape ensured the sufficient intracellular drug concentrations, which built foundation for improved efficacy of NNRTIs.
The enhanced endocytosisof T1144-NP-14fby the nanoparticle drug carrier resulted in a significant increase of anti-HIV-1 potency. The inhibitory activity of T1144-NP-14fagainst infection of HIV-1 IIIB and Bal was 25.12-and 40.13-fold higher than that of T1144 peptide, respectively, and 20.95-and 36.03-fold higher than that of 14f solution (Fig. 4cand 4d). One possiblemechanism underlying thisincreased antiviral activity could be attributed to the interaction betweenT1144 peptide and thegp41 NHR-trimer to block6-HB formation,thus avoidingviral entry and subsequent infection of the target cells.On the other hand,14fplayeda decisive role in reverse transcriptase inhibition, as evidenced by ELISA (Fig. 4a)and cell-cell fusion (Fig. 4b)examination.
The In vitrocytotoxicity experiment showed that 14f solution and NPs exhibited no significant cytotoxicity to cells at the concentration of 14f or T1144 as high as 4000 nM (Fig. S3, ). These data indicated that enhanced internalization of T1144-NP-14f in MT-2 and M7 cells did not cause cytotoxicity, suggesting that PEG-PLA-based nanomaterials can be considered as safe polymer drug carriers for human use.
In vitrorelease of 14f was evaluated in PBS (pH 7.4) that showed an apparent biphasic release pattern for T1144-NP-14f and NP-14f. The release of 14f from T1144-NP-14f was significantly slower than that of 14f solution. The initial faster burst release was believed to derive from the agents that located at the outer layer of the particles, while the later and slower release appeared to result from agents affected by erosion or degradation of the matrix [47]. The pharmacokinetics profile of the T1144-NP-14f, NP-14f, and 14f formulations was studied on SD rats after intravenous administration. The 14f formulation exhibited quicker blood clearance, shorter half-life and lower AUC0-tfrom systemic circulation, while T1144-NP-14f and NP-14f showed significantly prolonged elimination half-life, decreased clearance rate, and extended AUC 0-24h, suggesting that the nanocarrier was effective in facilitating the circulation of 14f molecules and that T1144 peptide did not change the circulation behavior of T1144-NP-14f. We concluded from these findings that the PEG polymer coating on the surface of NPs could improve the in vivopharmacokinetics profile of T1144-NP-14f.
The emergence of nanotechnology has made a significant impact on the development of clinical therapeutics, particularly providing an unparalleled opportunity for development of anti-HIV drugs. The application of nanotechnology offers the superiority of using anti-HIV drugs at lower dose, reduced systemic toxicity and enhanced therapeutic efficacy. However, translation of nanotechnology-based anti-HIV therapy into the clinics remains challenging. Significant efforts are needed to promote the translation process, including: 1) selection of absolutely biocompatible and biodegradable composition of nanoparticles; 2) carefully and rationally tailoring size and physicochemical property of the nanoparticles; and 3) providing desirable control over the drug biodistribution and pharmacokinetics to improve efficacy and reduce toxicity of nanoparticle-based anti-HIV therapeutics.
In conclusion, we employed biodegradable organic nanoparticles,PEG-PLA-NP, for encapsulation of anti-HIV-1 drug 14f and conjugation of T1144, a third-generation HIV-1 entry inhibitor, on the surface of nanoparticles. Our results demonstrated that the combination of entry inhibitor with reverse transcriptase inhibitor was highly effective in inhibiting R5-HIV-1 and X4-HIV-1, primary HIV-1 isolates, NNRTI-resistant HIV-1 strains, and T1144-resistant HIV-1 strains. Additionally, T1144-NP-14f exhibited enhanced intracellular uptake, sustained controlled release behavior and prolonged blood circulation time. The results indicated that the new cocktail-like nanoparticle drug delivery platform could serve as an effective anti-HIV-1 regimen by taking advantage of extrinsic and intrinsic antiviral activity of the partner drugs.
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