Research paper
pH-responsive delivery of Griffithsin from electrospun fibers

https://doi.org/10.1016/j.ejpb.2018.04.013Get rights and content

Abstract

Human immunodeficiency virus (HIV-1) affects over 36 million people globally. Current prevention strategies utilize antiretrovirals that have demonstrated protection, but result in antiviral resistance, adverse toxicity, and require frequent administration. A novel biologic, griffithsin (GRFT), has demonstrated outstanding safety and efficacy against laboratory and primary HIV isolates and against intravaginal murine herpes simplex virus 2 (HSV-2) challenge, making it a promising microbicide candidate. However, transient activity and instability remain concerns surrounding biologic delivery, particularly in the harsh environment of the female reproductive tract (FRT). Recently, electrospun fibers (EFs) have demonstrated promise for intravaginal delivery, with the potential to conserve active agent until release is needed. The goal of this study was to fabricate and characterize pH-responsive fibers comprised of poly(lactic-co-glycolic acid) (PLGA) or methoxypolyethylene glycol-b-PLGA (mPEG-PLGA) with varying ratios of poly(n-butyl acrylate-co-acrylic acid) (PBA-co-PAA), to selectively release GRFT under pH-conditions that mimic semen introduction. Fibers comprised of mPEG-PLGA:PBA-co-PAA (90:10 w/w) demonstrated high GRFT loading that was maintained within simulated vaginal fluid (SVF), and pH-dependent release upon exposure to buffered and SVF:simulated semen solutions. Moreover, GRFT fibers demonstrated potent in vitro efficacy against HIV-1 and safety in vaginal epithelial cells, suggesting their future potential for efficacious biologic delivery to the FRT.

Introduction

Sexually transmitted infections (STIs) affect hundreds of millions of people globally, with more than one million new infections each day [1]. Infection with human immunodeficiency virus (HIV) has become increasingly common in women, with disproportionate rates seen in minority populations [2]. In addition, the majority of HIV-infected women also experience co-infection with herpes simplex virus type 2 (HSV-2), which impacts over 500 million people [3]. HSV-2 infection has been shown to significantly enhance HIV acquisition by as much as 2 to 7-fold [4], [5]. Correspondingly, the challenges in HSV-2 prevention and treatment, combined with this high global incidence and propensity for co-infections, contribute to the need for multipurpose platforms that prevent both HSV-2 and HIV infections. With no cure currently in place, there is a crucial need to develop a safe and effective prevention method against a variety of STIs including HIV and HSV-2.

To date, oral and topical pre-exposure prophylaxis (PrEP) have relied primarily on antiretroviral (ARV) drugs to inhibit infection – while lacking the integration of biological agents. Furthermore, traditional oral and topical PrEP approaches have often focused on single indication products that have demonstrated efficacy against one type of viral infection, necessitating a combination of two or more agents in one therapy. The development of a platform that can prevent one or multiple viral/bacterial infections, while also increasing user compliance via a prolonged- or inducible-delivery platform, is urgently needed [6], [7], [8], [9], [10], [11]. However, continued challenges of PrEP include the need for lifelong daily user adherence; potential renal and bone toxicity; associated decreases in condom use; and the long-term development of ARV resistance [12], [13]. Moreover, to date, few biological agents have been explored to mitigate infection.

To address these multipurpose needs, an antiviral lectin, Griffithsin (GRFT), is currently being developed as a potent entry inhibitor against a variety of infection types. Originally identified and purified from the red alga Griffithsia sp. [14], GRFT is considered a promising microbicide candidate to prevent the sexual transmission of HIV. Of both synthetic drugs and biologically-based inhibitors, GRFT has potent anti-HIV activity in the picomolar range against both laboratory and primary isolates of HIV, and inactivates HIV-1 almost immediately upon contact [15]. Additionally, a 0.1% GRFT gel has demonstrated protection in mice against intravaginal HSV-2 challenge, demonstrating its potential against both HSV-2 and HIV-1 infections [16]. Moreover, GRFT has an excellent safety profile [14], [17], [18], [19], [20], negligibly induces pro-inflammatory cytokines [20], [21], and has demonstrated synergy with ARV agents [22], suggesting the benefits of future co-administration strategies. Finally, GRFT is being developed as a low-cost alternative with the ability to manufacture in large-capacity and possesses environmental stability, prompting its current testing in clinical trials [15], [16].

Despite the promise of GRFT and other microbicide candidates, significant hurdles face the clinical delivery of many active agents. A common theme, broadly and irrespective of application, is that existing topical dosage forms often necessitate daily administration; have low intravaginal residence time; and contribute to messiness via leakage. These issues adversely impact both user adherence and efficacy [6], [7], [8], [9], [10], [11] and often provide only transient prophylactic or therapeutic benefit [23], [24]. In particular, vaginally-administered gels are challenged with leakage from the vaginal cavity and remaining present to deliver active agents [25], [26], [27]. While, intravaginal rings offer longer delivery durations and durability [28], the high temperature processing conditions often associated with fabrication may prove challenging for the incorporation of biologics. Additionally, the administration of biological active agents may necessitate new considerations for delivery, to provide sustained- or inducible-release and protection of more rapidly degradable biologics. Given these challenges, next-generation topical formulations should be convenient and easy to administer, inexpensive to produce, and provide stability for and efficacy of incorporated agents for an appropriate time frame surrounding coitus.

Recently, polymeric electrospun fibers (EFs) have been investigated as a new delivery platform for reproductive applications, demonstrating both on-demand and sustained protection against HSV-2 and HIV-1 infections [29], [30], [31], [32], [33], [34]. However, one of the challenges of delivery vehicles, including EFs, is that to provide adequate protection they must release therapeutically relevant concentrations of active agents for the duration of use. This often requires frequent administration and highly localized doses to maintain adequate release for prolonged durations. While user adherence may be increased by developing a product that necessitates less frequent application, designing a dosage form that is efficacious regardless of administration time is challenging. Many sustained-release formulations undergo a “burst” release phase, where a significant fraction of active agent is released within the early hours of delivery – regardless of whether this time frame is suitable for protection [35].

An alternative approach is to design a product that requires less frequent dosing, by inducing the release of active agent only when needed, in response to microenvironmental cues. This strategy has the potential to conserve active agent from unnecessary release, provide protection independently of administration time, and deliver active agents directly to the target site of virus entry. One such cue in the reproductive tract, increased pH, is associated with semen infiltration peri- and post-coitus. While the “normal” vaginal pH ranges from 4.0 to 5.0, exposure to semen (pH ∼ 7.5) increases the local pH to more neutral levels. We expect a pH-responsive delivery vehicle that responds to increases in intravaginal pH, will only release active agent when triggered by semen, while maintaining the bioactivity and payload of encapsulated biologics under non-coital conditions.

While pH-responsive delivery has been used in a variety of drug delivery applications [36], [37], [38], [39], [40], [41], [42], thus far pH-responsive dosage forms are in the early stages of development for delivery to the female reproductive tract (FRT) [27], [39], [43], [44], [45], [46]. Prior to the use of electrospun fibers for intravaginal applications, temperature and pH sensitive hydrogels were developed to impart the dual advantages of semen-triggered release and vaginal distribution and retention prior to intercourse [27]. Hydrogels with pH-responsive properties have been shown to release effective concentrations of antivirals. However, hydrogels tend to provide more transient protection due to their propensity for leakage from the FRT. Similarly, polymeric NP platforms comprised of poly(lactic-co-glycolic acid) (PLGA) and S-100 Eudragit® blends were evaluated to provide pH-responsive release of the antiretroviral reverse transcriptase inhibitors, tenofovir and TDF [43]. Increased S-100 ratios resulted in decreased encapsulation efficiency, while conversely providing improved pH-dependent release. Similar studies assessed the mucosal delivery of pH-sensitive Eudragit S-100 NPs loaded with hydrophilic or hydrophobic molecules [39], demonstrating retention of molecules within NPs under acidic intravaginal pH and releasedupon exposure to more neutral pH conditions. This study additionally demonstrated the uptake and biocompatibility of NPs in vaginal cells [39]. Most recently, spray dried mucoadhesive and pH-responsive TFV microspheres prepared from polymethacrylate salts were fabricated, resulting in ∼90% release within the first hour, while demonstrating biocompatibility and mucoadhesivity to vaginal cells and porcine vaginal tissue [44].

Relative to gel and NP delivery platforms, electrospun fibers have recently emerged as an alternative intravaginal delivery platform that offer a durable stationary reservoir of encapsulated agents. However, many of these studies have focused on the delivery of antibiotics or ARVs, relative to new biologics [39], [43]. One of the first studies to investigate pH-responsive fibers for vaginal applications, demonstrated that cellulose acetate phthalate (CAP) fibers highly incorporated the reverse transcriptase inhibitors etravirine and TDF, and the hydrophilic dye rhodamine [46]. The CAP polymer, itself a potent antiviral, is minimally soluble in acidic conditions, and the addition of SSF rapidly dissolved the CAP fibers, releasing the encapsulated drugs. While this quick degradation was attributed to the natural (vs. synthetic) polymer chemistry, the fiber degradation raised concerns over long-term structural integrity as well as corresponding protection, prompting the development of fibers with improved mechanical properties. To address this need, coaxial fibers, comprised of a polyurethane core and CAP shell layer, were fabricated to provide pH-inducible release of rhodamine, while demonstrating enhanced mechanical properties [47]. Finally, fibers comprised of Eudragit L-100 encapsulating horseradish peroxidase and alkaline phosphatase were fabricated using emulsion electrospinning [48]. These fibers modulated protein release in response to pH, while preserving protein activity. In another study, pH-responsive fibers comprised of poly(methacylic acid-co-methyl methacrylate), encapsulating the ARVs, dapivirine and etravirine, were fabricated [49]. These fibers demonstrated sustained-release of therapeutics within acidic conditions, while the fibers rapidly dissolved in alkaline pH, to provide encapsulant release.

While a variety of pH-responsive platforms have demonstrated promise against STIs, many of these platforms, inclusive of electrospun fibers, have focused on the delivery of antibiotics or ARVs, relative to new biologics [39], [43]. Recently, we and others have developed EFs as an efficacious platform to provide sustained-delivery of antiviral drugs to the FRT [29], [30], [31], [32], [33], [34]. Building upon this work, the goal of this project was to develop and test pH-responsive EFs that incorporate the antiviral lectin, GRFT. Griffithsin fibers were designed to address the needs of an on-demand delivery system, while providing a delivery vehicle that may reduce the frequency of daily administration. It is well known that poly(acrylic acid) (PAA) has been used to fabricate a variety of pH-responsive dosage forms [50], [51]. Moreover, due to its carboxylic acid groups that are deprotonated within acidic environments (here, vaginal), active agents are retained under slightly acidic conditions. Conversely, in neutral and alkaline environments, the carboxylic acid groups become ionized, inducing electrostatic repulsion, which results in fiber swelling and agent release into the surrounding medium [51]. Additionally, PAA as well as the polymer poly(n-butyl acrylate) (PBA) have been used to produce mucoadhesive polymers, demonstrated in buccal delivery and other applications [52], [53], [54], [55], [56], [57]. Given these properties, we selected the copolymer, PBA-co-PAA, to blend with known sustained-release polymers, PLGA and methoxypolyethylene glycol (mPEG)-PLGA, to provide pH-dependent GRFT release. We hypothesized that the encapsulated GRFT released from these pH-responsive fibers would retain antiviral properties relative to free GRFT and that utilizing PBA-co-PAA fibers to deliver biological entry inhibitors, such as GRFT, may prove useful to conserve the payload and activity of active agent when needed.

Section snippets

Materials

Carboxyl-terminated poly(d, l-lactic-co-glycolic acid) (PLGA, 50:50, 0.55–0.75 dL/g, 31–57 kDa MW) was purchased from LACTEL® Absorbable Polymers (Cupertino, CA, USA). Methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA, 5,000:55,000 kDa) was obtained from PolySciTech® Akina Inc. (West Lafayetter, IN, USA). Poly(n-butyl acrylate-co-acrylic acid) (PBA-co-PAA, 50:50, catalog number 19911-10), an alkali-soluble 20% latex in alcohol was purchased from Polysciences Inc.

Fiber morphology

The morphology and microstructure of PLGA and mPEG-PLGA fibers were assessed with scanning electron microscopy (Fig. 2). In addition, the morphology of PLGA and PBA-co-PAA polymer fibers with blend ratios of: 100:0, 90:10, 85:15, 80:20 and 75:25 and mPEG-PLGA:PBA-co-PAA (90:10) fibers were evaluated (Fig. 3). As shown in Fig. 2, Fig. 3, all formulations provided well-defined fiber morphologies. The fiber diameters of the different polymer blends tested for pH-responsive release were assessed

Discussion

While previous work has demonstrated the potential to provide pH-responsive release of traditional antivirals, here we present a method to induce the pH-responsive release of the antiviral lectin, GRFT. Griffithsin is a promising new biologic for use against HIV, HSV-2, human papillomavirus, and a variety of other viruses, due to its potent binding and antiviral activity [15], [17], [65], [66], [67]. Additionally, GRFT has demonstrated stability and safety, prompting its development in clinical

Conclusions

Drug delivery systems in which active agent release can be tailored to release in response to incoming stimuli are particularly promising for biologics that may lose activity quickly and be expensive to produce. In this study, pH-responsive fibers comprised of PLGA, mPEG-PLGA, and PBA-co-PAA polymer blends were fabricated to provide pH-responsive release of GRFT. Of the formulations tested, the mPEG-PLGA:PBA-co-PAA (90:10) blend provided the optimal release of GRFT, exhibiting increased release

Acknowledgement

We are grateful for funding from the Jewish Heritage Fund for Excellence, the James Graham Brown Cancer Center, and the National Center for Research Resources CoBRE (1P30GM106396) to conduct this work.

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