Antiretroviral drug-loaded nanoparticles-in-films: a new option for developing vaginal microbicides?

KEYWORDS:

• HIV
• microbicides
• nanomedicine
• pharmacokinetics
• pre-exposure prophylaxis

1. Introduction

HIV/AIDS remains a global health challenge requiring more investment in prevention. Vaginal microbicides are antiretroviral drug-based products intended to be locally administered in order to prevent HIV transmission at the mucosal level [1]. So far, only a tenofovir (TFV) gel and a dapivirine (DPV) ring were shown safe and able to reduce HIV acquisition by women in clinical trials [2,3]. However, protection from infection was only modest: reduction in overall HIV incidence ranged between 27% for the ring and 39% in the case of the gel. Failure to completely prevent viral transmission was largely associated with erratic adherence and/or product misuse, which led to poor genital pharmacokinetics (PK). Indeed, the relationship between local PK and pharmacodynamics (PD) has been fairly well established for TFV and DPV [4,5]. Although threshold drug concentrations conferring full protection may be difficult to define, it seems apparent that higher drug levels are associated with lower risk of infection. For instance, cervicovaginal fluid concentrations of TFV of at least 1000 ng/mL have been associated with 76% risk reduction, but dropped to 65% when the lower limit was set at 100 ng/mL [5]. This increasing evidence of the role of PK/PD in defining the efficacy of microbicides seems to justify research into novel strategies that are able to improve drug distribution and retention in the vagina. The application of nanotechnology principles in the design of microbicides has been explored over recent years [6]. Potentially advantageous features include the ability to improve drug solubility, control drug release, protect labile active compounds, affect drug safety and activity, enhance genital drug distribution and retention, and optimize the interaction of drugs with HIV and/or particular cell populations. Nano-microbicides may be conveniently classified as nanosystems with inherent activity or nanocarriers for active molecules. Dendrimers are particularly relevant in the case of the former and have been extensively studied [7]. As for drug nanocarriers, polymeric nanoparticles (NPs) are in the forefront, mainly for the delivery of potent antiretroviral drug candidates. In different cases, animal studies have been able to show that the association of drugs to NPs can enhance genital PK [8] or even protect against vaginal HIV transmission [9].

Despite all efforts made so far, important issues remain to be addressed, namely the design of platforms that allow suitable administration of microbicide nanosystems. Natural candidate dosage forms include those previously used for microbicide drug delivery. Gels are in the forefront but technical problems, namely related with premature drug release from nanocarriers, and user-related issues (e.g. vaginal leakage and discomfort) may restrain their usefulness. Such limitations could be overcome by solid systems such as rings and tablets but concerns exist regarding suitable incorporation and release of nanosystems. Polymer-based thin films have also been considered for the vaginal administration of various microbicide drugs [10] and could serve the purpose of delivering nanosystems. We hereby discuss relevant features and potential of films to incorporate and aid in the vaginal delivery of promising antiretroviral drug-loaded NPs, further referred to as NPs-in-films.

2. Nanoparticles-in-films as potential vaginal microbicide products

2.1. Technology and potential

Polymeric films have long been considered as suitable and highly acceptable dosage forms for the vaginal delivery of drugs. These are typically thin, soft, flexible, and transparent/translucent rectangular- or square-shaped sheets obtained by solvent-casting or, less frequently, hot-melt extrusion. Apart from active molecule(s), matrix-forming polymers and plasticizers, other components may also be present (e.g. preservatives, stabilizers or disintegrants). Research and development have been prolific in the case of vaginal microbicide films [10]. Films are typically designed to disintegrate/dissolve within a few minutes upon contact with vaginal fluids, thus allowing for drug(s) to be quickly released. Advantageous features include good physicochemical stability and resistance from microbial contamination (often abbreviating the use of preservatives), reduced weight and volume favoring portability, on-demand and discrete use, ability to be self-inserted without requiring an applicator, and avoidance of leakage after administration. On the downside, vaginal administration may be troublesome since films will start disintegrating/dissolving immediately upon contact with aqueous environments [10].

There are several potentially interesting features for associating drug-loaded NPs and films. Incorporation itself may be conveniently performed during manufacturing by solvent-casting. This last technique typically involves dissolving or dispersing film components in an aqueous solvent, which is then cast onto molds or other appropriate media. Films are obtained upon drying and can be further cut into smaller pieces of adequate shape and size. The incorporation of drug-loaded NPs can be conveniently performed immediately before casting and drying of films. One limiting step, however, is related with the possibility of premature drug release from nanocarriers during drying, while solvent content is high. If possible, abbreviating the amount of time required for this step may be useful, namely by increasing temperature, promoting air renewal or reducing atmospheric pressure, decreasing the amount of solvents used, or using non-solvents for drugs being used. Combining two or more of these strategies and using specialized equipment should be considered. Hot-melt extrusion seems of limited application to most drug nanocarriers being studied due to high processing temperatures required for melting film-forming polymers and other film components. Another possibility involves the association of NPs to pre-prepared films. However, consistent and uniform application of nanosystems onto the surface of films (e.g. using a spraying system) may be challenging. One other important limitation may be the ability to incorporate only relatively low amounts of nanosystems before the original characteristics of films being compromised.

2.2. Recent developments

Various research groups are currently engaged in developing microbicide drug-loaded NPs-in-films. For instance, Srinivasan et al. [11] have reported on a NPs-in-film formulation for the delivery of IQP-0528, an investigational non-nucleoside reverse transcriptase inhibitor. A poly(vinyl alcohol) (PVA)/hydroxypropyl methylcellulose (HPMC)-based system containing IQP-0528-loaded poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA):Eudragit® S-100 NPs (mean diameter of 434 nm) was manufactured by solvent-casting and specifically designed to quickly dissolve upon administration. When tested intravaginally in Macaca nemestrina, NPs-in-film did not significantly affect local PK as compared to a similar film containing dispersed IQP-0528. However, values around 1–5 logs greater than the in vitro 90% inhibitory concentration for the drug were observed up to 24 h post-administration, thus suggesting the potential for protecting against HIV transmission.

Our research group has also recently proposed a PVA/HPMC film for the administration of TFV-loaded PLGA-based NPs (mean diameter of 127 nm) [12]. NPs were incorporated in high amounts (over 50% of the total weight of the final film), thus inducing significant changes to the mechanical and biophysical properties of plain films. The utilization of an aminated lipid stearylamine in the composition of NPs to improve drug association led to significant increase in cytotoxicity. However, no safety issues were reported when TFV-loaded NPs-in-film were administered intravaginally to mice for 14 days. A possible explanation for these apparently divergent in vitro/in vivo outcomes may be related to the adsorption of PVA and HPMC at the surface of NPs, thus abbreviating deleterious interactions with the mucosa.

NPs-in-film systems may also be particularly appealing for the delivery of labile molecules. Gu et al. [13] proposed a quick dissolving PVA/lambda-carrageenan-based film as platform for the delivery of targeted, siRNA-loaded polymeric NPs. PEG-PLGA-based NPs were used for associating complexes of polyethylenimine (PEI):siRNA against SNAP-23 (a host protein involved in the exocytosis of HIV). Particles were also surface functionalized with anti-HLA-DR antibody for enabling targeting to HLA-DR+ dendritic cells. These NPs (mean diameter of 232 nm) were further incorporated into films during production by solvent-casting. Films were only evaluated in vitro but obtained data using a co-culture model of vaginal epithelial VK2/E6E7 cells with underlying mKG-1 dendritic cells suggest that the system was successful in allowing mucosal penetration and selective intracellular delivery of siRNA to dendritic cells, resulting in effective SNAP-23 silencing.

Current opinion points out to the need of developing combination microbicide products. However, incorporating compounds featuring distinct physicochemical properties, namely regarding solubility, may pose considerable challenges. Our group recently developed a PVA/HPMC-based film incorporating plain hydrophilic TFV and hydrophobic efavirenz (EFV) associated to PLGA NPs with mean diameter of 275 nm (TFV/EFV-NPs-in-film) [14]. Obtained films (Figure 1) were fully characterized for mechanical, biophysical, and in vitro biological features and shown suitable for vaginal use. Moreover, TFV/EFV-NPs-in-film (5 × 5 mm) were able to affect local PK in CD-1 mice. Although larger animals may seem more appropriate for such studies, the vaginal administration of films to mice is easy and convenient to perform, and this species, namely humanized models, may be suitable for efficacy studies using HIV-1. The use of TFV/EFV-NPs-in-film increased vaginal residence of EFV as compared to the film containing EFV directly dispersed in the film matrix (TFV/EFV-film). For example, values of area-under-the-curve between 15 min and 24 h were around 2.2- and 5.7-fold higher in vaginal lavages and tissues, respectively, for TFV/EFV-NPs-in-film. Values for TFV were not affected, which could be foreseen due to the fact that this drug was simply incorporated into the film matrix. Also, systemic exposure to EFV and TFV was low in all cases and no safety issues were noticed. Overall, data seem to support that NPs-in-film may be suitable systems for delivering intravaginally microbicide drugs with different solubility profiles and provide allegedly protective levels for longer periods. These assumptions, however, require in vivo efficacy studies to be conducted in order to truly assess the potential of TFV/EFV-NPs-in-film.

Figure 1. Selected features of TFV/EFV-NPs-in-film. (a) Photograph and (b, c) SEM micrographs (surface and exposed side sections after fracture; bars = 50 µm) of TFV/EFV-NPs-in-film. (d) EFV release profile in simulated vaginal fluid from TFV/EFV-film, EFV-NPs (i.e., without being incorporated into film) and TFV/EFV-NPs-in-film. Relevantly, almost half of the drug content was released within 1 h, which could provide a burst dose for immediate protection, while continuously drug release up to 24 h may aid sustaining protection. Points and vertical bars in drug release profiles represent mean values and standard deviations, respectively (n = 3). Please note the square root of time in the x-axis. Modified from [14], Copyright (2016), with permission from Elsevier.

The development of NPs-in-film for the vaginal delivery of antiretroviral drugs is still in its infancy and many possibilities remain to be explored. Increasingly elegant solutions may likely emerge from current research in the field of biomedical engineering as alternatives to classical solvent-casting. An example may be the incorporation of NPs in polymer-based fibers that are then shaped as film-like products. No definitive experimental work on such type of system is available but a preliminary report by Krogstad et al. [15] evidenced the feasibility of incorporating etravirine-loaded PLGA NPs into PVA electrospun fibers. These last were shaped as white mat-like thin sheets and shown to affect local PK after intravaginal administration to mice.

3. Expert opinion

There is still lack of knowledge regarding which platforms may allow suitable formulation of microbicide nanosystems into vaginal products that can be used by women. Polymeric films are starting to be considered for such purpose but data so far suggest that NPs-in-films may impact important features such as vaginal PK. The stability provided by their solid state nature as well as the versatility to incorporate various active molecules are also advantageous characteristics. Further steps into the full development of NPs-in-films include the investigation on improved and scalable manufacturing methods that can overcome changes to the original properties of nanocarriers, namely regarding drug release during drying stages, and the release kinetics of nanosystems upon film disintegration. Understanding drug release from NPs-in-films and the individual contributions of nanocarriers and film matrices (either in parallel or tandem) is an important matter that requires to be addressed. Classical in vitro drug release assessment is insufficient and innovative techniques are needed. Feasibility and affordability of manufacturing scale-up are likewise relevant issues although the extension of previous work on films containing NPs for oral application already provides good hints for abbreviating several problems. Additionally, studies concerning changes to the surface of released nanosystems due to the adsorption of other film components and their role in the biological performance of such systems are required. Finally, expanded animal safety and efficacy testing are necessary in order to definitively understand the potential of antiretroviral drug-loaded NPs-in-films before considering clinical testing.

Declaration of interest

J das Neves and B Sarmento are co-inventors of one Portuguese patent application (20151000087553, Instituto Nacional da Propriedade Intelectual, Lisbon, Portugal) and one international patent application (PCT/IB2016/056695, World Intellectual Property Organization, Geneva, Switzerland) related with NPs-in-films. B Sarmento is principal investigator of a project for the development of NPs-in-films funded by Programa Gilead GÉNESE, Gilead Portugal (ref. PGG/046/2015). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

This work was financed by Programa Gilead GÉNESE, Gilead Portugal (ref. PGG/046/2015), by Fundação para a Ciência e a Tecnologia (FCT), Portugal (VIH/SAU/0021/2011), by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT/Ministério da Ciência, Tecnologia e Inovação in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274). José das Neves gratefully acknowledges FCT for financial support (SFRH/BPD/92934/2013).