**5.2. The 'shock and kill' strategy**

Upon systemic administration, most of the nanoparticles are instantaneously opsonized with plasma proteins and rapidly cleared off from circulation by the phagocytosis. The macro‐ phages of the reticulo-endothelial system are the principal cells which are involved in the degradation and clearance of the nanoparticles [118]. This forms the basis of passive targeting which aims at achieving high concentrations of anti-retroviral agents in the reticulo-endothe‐ lial reservoir as a consequence of phagocytosis of the drug loaded nanoparticles. Studies have demonstrated the achievement of higher drug concentrations in the reticulo-endothelial cells following the administration of liposomes, nanocapsules or polymeric nanoparticles loaded with anti-retroviral drugs [119]. Likewise, passive targeting of the lymphatic reservoir can be achieved by incorporation of the nanoparticles with lipids such as phosphatidylcholine and cholesterol or by surface coating of the nanocarriers with polyethylene glycol [120, 121].

224 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

Apart from passive targeting, specific drug delivery to the anatomical reservoirs can be accomplished by active targeting strategies. Nanocarriers tagged on their surface with galactose or mannose residues or anti-HLA-DR monoclonal antibodies, effectively localize in the reticulo-endothelial system which have abundant receptors for these ligands such as the galactose and lectin receptors and the HLA-DR determinant of MHC-II respectively. Active targeting of the cellular reservoirs is based on the aforementioned principle of employing nanocarriers with homing ligands to viral components on infected cell surface. The infected resting CD4 T-cells containing HIV envelope glycoproteins on their surface attract the drug loaded nanoparticles coated with recombinant CD4 molecules or Fab fragments of monoclonal

Nanotechnology also offers a solution for the problem of poor drug penetration in certain anatomical sites. One such anatomical reservoir is the brain, where the infected microglial cells rest safely with the protection conferred by the blood brain barrier against the system‐ ically administered anti-retroviral agents. The blood brain barrier functions not only by preventing the permeation of the circulating anti-retroviral compounds into the brain tissue, but also by efflux of a considerable portion of the compounds that have managed to cross through. With the ability to increase the crossing and reduce the efflux of drugs, nanotechnol‐ ogy methods help in reservoir elimination by overcoming the privilege offered by the blood

Nanotechnology methods that increase the drug transport across the blood brain barrier function by mimicry of natural substrates, utilization of cell penetrating peptides or by active targeting of molecules of abundance in neuronal vasculature with suitable surface ligands. The non-ionic surfactant 'polysorbate-80' has found to be an effective enhancer of drug delivery to the brain using the principle of substrate mimicry. Upon systemic administration, the drug loaded nanoparticles coated with polysorbate-80 adsorb various apolipoproteins in circulation to form a complex that mimics lipoproteins. Presuming them as natural substrates, the blood brain barrier permits the entry of these complexes by receptor mediated transcytosis resulting in delivery of the drug to the brain tissue [123]. Nanoparticles coated with cell penetrating peptides such as the HIV-1 tat peptide has shown enhanced efficacy in crossing the blood brain barrier. As the microvasculature of the brain is rich in receptors such as transferring receptor,

anti-gp120 antibodies [13].

brain barrier [122].

Considered as the 'holy grail' of HIV eradication research, this strategy aims at depletion of the latent reservoirs by widespread activation of the provirus using 'shocking agents' and subsequent killing of the actively replicating virions. Reactivation of the quiescent viral genes can be achieved either by inactivating the repressing factors and/or by activating the tran‐ scription factors [13].

Deacetylation and methylation of histones and DNA methylation are the few processes that have been identified to favour the proviral repression and hence the inhibitors of these processes are considered as attractive candidates for shocking agents. Inhibitors of the histone deacetylase group of enzymes; valproate and vorinostat have been tried out for viral reacti‐ vation with limited success [124]. Congeners of vorinostat such as givinostat, belinostat, panobinostat and droxinostat and other novel histone deacetylase inhibiting compounds such as oxamflatin, romidepsin are being evaluated for their efficacy in reactivating viral replication. Histone methyltransferase inhibitor chaetocin and DNA methyltransferase inhibitors azacy‐ tidine and decitabine are also being assessed for their efficacy as shocking agents [125].

Provirus could also be drawn into active replication by making the intracellular milleu conducive for transcription [126, 127]. This could be achieved by activating the transcription factors such as the NF-κB and transcription elongation factor- b. Initial studies that employed NF-κB activators such as interleukin-2 and monoclonal anti-CD3 antibodies failed to relieve the provirus from repression. This led to the identification of a gatekeeper kinase which has to be concurrently activated along with NF-κB to facilitate viral replication. Other compounds such as prostratin and bryostatin-1 are also being evaluated for efficacy in the activation of NF-κB pathways [128]. Activators of elongation factor-b such as hexamethylene bisacetam‐ mide, disulfiram and certain quinolones are being evaluated as shocking agents. Apart from these, interleukin-7, agonists of Toll like receptor-9 and novel syenthetic molecules have been identified to activate viral replication in the latent reservoirs [125, 129]. All of these transcrip‐ tion activators are being assessed for stand-alone use or for use in combination with inhibitors of proviral repression.

None of the agents of this attractive strategy has provided satisfactory results so far. The dosing and combinations have to be worked out to extract maximum effects out of these shocking agents. As most of the shocking agents are related to anti-cancer drugs, they are likely to have a significant adverse effect profile. The activators of the NF-κB pathway are notorious for triggering a plethora of cytokines and can cause a fatal cytokine storm if administered at higher concentrations. Another important drawback is that, even if an efficient shocking agent is identified, killing of the actively replicating virions depends on anti-retroviral drugs which are expected to lose their efficacy in the future due to the evolution and dissemination of resistant viral population. Nevertheless, this strategy has immense potential in eradicating viral reservoirs if the various setbacks are addressed over time [130].

Apart from the trial of different compounds, techniques of gene therapy have also been tried out to activate viral reservoirs with limited success. Herpes virus and lentiviral vectors loaded with genes coding for key viral proteins involved in replication were found to induce repli‐ cation and release of the provirus from the latent CD4 T-cells [131]. Problems with *in vivo* administration, lack of specificity in targeting the cellular reservoir, low frequency of recom‐ bination are the various setbacks which have withheld the progress of this technology in reservoir elimination [132].
