The HIV-1 pandemic continues to grow. An estimated 33.2 million people globally are infected with the virus; 2.2 million deaths occurred in 2008 alone, with the majority coming from the resource-limited areas [15
]. The requirement for uninterrupted daily dosing, suboptimal patient adherence and inadequate tissue penetration continue to lead to treatment failure in a proportion of patients receiving ART [1
]. Thus, our research effort in ART delivery has focused on developing long-acting parenteral drug formulations that can be maintained inside cells for periods measured in weeks and that travel specifically to viral sanctuaries [17
Using poorly water-soluble ART, we manufactured nanosuspensions by homogenization and/or sonication techniques, and studied, in a complete manner, the physical properties of nanoART including size, charge and surface coating. We now demonstrate that we are able to directly modify the pharmacokinetics and cellular handling of drugs by altering the physical properties of NP. Most importantly, we demonstrated proof of concept in that cells pretreated with nanoART release the drug and inhibit HIV-1 infection for up to 15 days after treatment.
Macrophage-based nanomedicine delivery has a number of advantages for ART in an infected human host, including drug stability, delivery into viral sanctuary sites and sustained release. First, oral administration of drugs shows limited biodistribution and commonly results in the development of viral sanctuaries. This can increase the rate of viral mutation, affect drug resistance and result in treatment failure [2
]. In addition, inadequate penetration of ART drugs into the genital tract raises concern about the ongoing risk of transmission, even when plasma HIV RNA levels are below the limit of detection [28
]. Second, once the NPs gain entry into the monocyte or MDM, the cells can hold the drug in an active and stable form for periods of weeks. As such, they could provide a means to improve dosing schedules, adherence to therapy and the therapeutic index. Third, since the very same cell that carries the virus throughout the body is being used to deliver drug, it is likely that suitable drug concentrations could reach tissue sites that otherwise would have little or no drug penetration. Indeed, we posit that such a drug-delivery system using highly stable nanoART specifically targeted to monocytes and macrophages could greatly improve therapeutic outcomes.
These studies are but an incremental advance from what is being realized for nanomedicine. Indeed, nanotechnology has revolutionized pharmacology and drug delivery [30
]. NPs can be altered in size, shape and composition to allow incorporation of drugs with a variety of biochemical properties. In addition, formulating drugs as NPs can allow for cellular transport and delivery. MDM have been shown to be potential drug vehicles for uptake, transport and delivery of nanoART [17
]. Based on known limitations, a broad range of methods was previously employed to incorporate ART drugs into nanocarriers, including mannosylated gelatin, squalenoyl NP, fullerene cages and polymeric nanogels [33
]. However, in all cases, cellular uptake and release of the drug was either limited or complicated by toxicity. A recent report demonstrated that sustained plasma concentrations of ART could be achieved over weeks to months, but this required intramuscular or subcutaneous administrations [38
]. Indeed, in our own work, using intravenous routes of injection, the absolute level of cellular uptake and distribution hindered the system. Therefore, in vitro
treatment with nanoART followed by adoptive transfer was required [17
]. This poses a challenge concerning the use of nanoART in the clinic. In order to overcome this and other barriers, our laboratories have designed highly stable nanoART to specifically target monocytes and MDM, maximize cellular uptake and extend antiretroviral activity. Comparisons between results obtained in this cell-based system with in vivo
drug distribution are currently ongoing in our laboratories.
The successful application of depot-injected risperidone in clinical practice demonstrated the benefits provided by long-acting drug therapies [39
]. Risperidone is an example of a drug that successfully transferred from daily oral administration to a sustained depot injection. Similar to the present situation, depot injection of risperidone was a modification of a clinically accepted oral therapy. The use of this and other long-acting antipsychotic compounds improved dosing schedules, enhanced treatment adherence and improved treatment outcomes. We propose using nanoART for cell-based drug delivery to create a similar long-acting drug treatment for HIV infection. If nanoART could be manufactured to be stable, actively taken up by cells, transported into tissue and slowly released, then injectable long-acting antiretroviral nanoformulations, which we are in the process of developing, could be realized for translational studies.
Nanosized drug crystals coated with a mixture of surfactants enhanced cellular uptake and maximized time of drug release. By modifying the size, coating and charge of nanoART, we have determined important physical characteristics that greatly enhance the pharmacokinetics and cellular handling of nanoART. We determined that nanoART, which are approximately 1 μm in size, are taken up most rapidly. It has been shown that larger NPs are taken up in greater amounts by MDM compared with smaller particles [43
]. This could potentially be due to the mechanism of uptake. Particles 1 μm or greater in size are generally taken up by phagocytosis, as opposed to smaller particles 300 nm or less, which are taken in by a pinocytotic-like mechanism such as clathrin/caveolae-mediated uptake [44
We also determined that the surfactant used to coat nanoART greatly affects how cells handle them. It has been demonstrated that the coating of a NP can greatly affect cellular handling and stability [46
]. In this study, a combination of DSPE-mPEG2000
and P-188 was optimal for IDV and RTV. However, EFV uptake and release was much greater when it was encapsulated with PLGA. This demonstrates that there may be an optimal single or combination of surfactants for each ART drug that best maximizes uptake and release by cells. We also determined that positively charged nanoART are taken up better than negatively charged ones; although the difference is slight, this parallels previous findings [49
Most important was the demonstration of the antiretroviral potential of nanoART. Cells pretreated with nanoART were protected against viral challenge for up to 15 days. Not only can cells actively take up nanoART, but they can also maintain the particles for long periods of time and allow steady release of drugs in clinically significant amounts.