Name: fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections
Uploaded: 2017-10-31T14:00:00
Description: Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

We are grateful to the Jewish Heritage Fund for Excellence for funding this research. We thank Dr. Stuart Williams II&#160;for generously providing usage of the electrospinning system. We also thank Dr. Kenneth Palmer for providing us with Griffithsin. We additionally thank Dr. Nobuyuki Matoba and his lab for training us in the GRFT ELISA work.

1. Preparation and Fabrication of the Electrospun Fiber Scaffold
CAUTION: All work with solvents or polymer solutions should be performed in a chemical fume hood. Refer to material safety datasheet of each reagent before starting the protocol.
<ol>
	<li>To electrospin a 3 mL 15% w/w PLGA polymer solution, weigh 720 mg of 50:50 poly(lactic-co-glycolic acid) (PLGA; 0.55 to 0.75 dL/g, 31-57 kDa) into a 10 mL scintillation vial. The volume of the solution is based on the typical batch s...

Due to their porous structures and large surface areas, EFs have found a variety of applications in healthcare, one of which includes serving as therapeutic delivery vehicles. Drugs and other active agents can be incorporated within EFs for tunable delivery, while biologics and chemical ligands can be conjugated to the fiber surface for cell-specific targeting52 or biosensing53. Here the fabrication of GRFT surface-modified PLGA EFs, as a delivery scaffold to prevent HIV in...

The use of EFs as a topical delivery platform has the potential to significantly reduce STIs. Currently, there are over 36 million people living with HIV, with over two million new cases of reported in 2015 alone1,2. Additionally, herpes simplex virus type 2 (HSV-2) infection affects hundreds of millions of people worldwide and has been shown to enhance the acquisition of HIV by 2 - 5 fold3. Due to this relationship between HSV-2 infection and HIV acquisition, there is significant interest in developing new active agents that provide simultaneous protection against multiple STIs. Moreover, the development of new vehicles to improve the delivery of these antiviral agents offers the potential to further enhance protective and therapeutic potency. Toward this goal, EFs have been investigated as a new delivery platform to reduce the prevalence of HIV-1 and HSV-2 infections.
During the past two decades, EFs have been extensively used in the fields of drug delivery and tissue engineering4. Often, biocompatible polymers are selected to easily translate to therapeutic applications. To fabricate polymeric EFs, the selected polymer is dissolved in an organic solvent or aqueous solution, depending on the degree of polymer hydrophobicity5. Active agents of interest are then added to the solvent or aqueous solution prior to the electrospinning process. The polymer solution is then aspirated into a syringe and slowly ejected while subject to an electrical current. This process typically results in polymer fibers with sheet or cylindrical macrostructures (Figure 1), and fiber diameters ranging from the micro- to nano-scale6. For most therapeutic applications, active agents are incorporated within the fibers during the electrospinning process and are released from the fiber via diffusion and subsequent fiber degradation. The rate of degradation or release may be altered by using different types of polymers or polymer blends to establish a desired release profile, imparting unique chemical and physical properties7, and promoting the encapsulation of virtually any compound. As such, EFs have proven beneficial to the delivery of small molecule drugs and biological agents including proteins, peptides, oligonucleotides, and growth factors6,8,9.
In the field of STI prevention, EFs have been recently used to incorporate and provide sustained- or inducible-release of antiviral agents10,11,12,13,14,15,16,17,18,19. In one of the earliest studies, pH-responsive fibers were developed to release active agents in response to environmental changes within the female reproductive tract (FRT), as an on-demand method of protection against HIV-111. Since, other studies have investigated polymer blends comprised of polyethylene oxide (PEO) and poly-L-lactic acid (PLLA), to evaluate the tunable release of antiviral and contraceptive agents for HIV-1 prevention and contraception in vitro12. Additional studies have demonstrated the feasibility of EFs to provide the following: prolonged release of small molecule antivirals14, strong and flexible mechanical properties20, 3-D delivery architectures21, inhibition of sperm penetration12, and the ability to merge with other delivery technologies13. Finally, previous work has evaluated polymeric fibers for the sustained-delivery of antiviral agents against common co-infective viruses, HSV-2 and HIV-114. In this study, polymer fibers provided complementary activity to antiviral delivery by retaining their structure for up to 1 month and providing a physical barrier to viral entry. From these results, it was observed that EFs may be used to both physically and chemically hinder virus infection.
While tunable release properties make polymeric EFs an attractive delivery platform for microbicide delivery, EFs have been developed in other applications to serve as surface-modified scaffolds7. EFs have been used to mimic the morphology of the extracellular matrix (ECM), often acting as scaffolds to improve cellular regeneration22, and enhance their utility in tissue engineering23,24. Fibers comprised of polymers such as poly-&#949;-caprolactone (PCL) and PLLA have been surface-modified with growth factors and proteins after electrospinning to impart ECM-like properties including increased cellular adhesion and proliferation25,26. Additionally, antimicrobial surface-modified EFs have been evaluated to prevent the growth of specific pathogenic bacteria27,28. Due to this versatility and the ability to induce biological effects, EF technology continues to expand across a variety of fields to provide multi-mechanistic functionality. Yet, despite their utility in a diversity of applications, surface-modified fibers have only recently been explored in the microbicide field29.
In parallel with the development of new delivery technologies to prevent and treat STIs, novel biological therapeutics have been developed. One of the most promising microbicide candidates is the adhesive antiviral lectin, GRFT30. Originally derived from a species of red algae, GRFT has demonstrated activity as a potent inhibitor of HIV, HSV-2, SARS, as well as Hepatitis C virus31,32,33,34,35,36. In fact, among biologically-based inhibitors, GRFT has the most potent anti-HIV activity, inactivating HIV-1 almost immediately upon contact30, while maintaining stability and activity in the presence of culture media from vaginal microbes for up to 10 days37. More recently, a 0.1% GRFT gel was shown to protect mice against intravaginal HSV-2 challenge, making it a promising candidate for the first line of protection against both HSV-2 and HIV-132,38. For HIV specifically, GRFT inhibits infection by physically binding gp120 or terminal mannose N-linked glycan residues on viral envelope surfaces to prevent entry38,39,40,41,42. This inhibition is highly potent, with IC50s approaching 3 ng/mL43. In addition to inhibiting HIV infection, studies have also shown that GRFT protects against HSV-2 infection by inhibiting the cell-to-cell spread of the virus32. In all cases, GRFT has been shown to be adhesive to viral particles, while demonstrating high resistance to denaturation. Last, GRFT has demonstrated synergistic activity with combinations of Tenofovir (TFV) and other antivirals44, making it feasible and likely beneficial to co-administer with EFs. The potent properties of GRFT make it an excellent biologically-based antiviral candidate, in which delivery may be enhanced with EF technology.
Utilizing this knowledge of the adhesive and innate antiviral properties of GRFT, a polymeric fiber scaffold was designed, that integrates these properties to provide the first layer of virus entry inhibition29. Finding inspiration in the way that cervicovaginal mucus hinders virus transport primarily through mucoadhesive mucin interactions, we hypothesized that by using EFs as a scaffold and covalently modifying the surface with GRFT, a high density of surface-conjugated GRFT would debilitate and inactivate virus at its entrypoint45,46,47. Here EFs were developed as a stationary scaffold to provide a protein-based, viral adhesive-inactivating barrier platform. We sought to combine the potent antiviral properties of GRFT with a biocompatible, modifiable, and durable polymer platform, to create a novel virus &#34;trap.&#34;
To achieve these goals, fibers comprised of PLGA were electrospun, and EDC-NHS chemistry was used to subsequently modify the EF surface with GRFT. PLGA served as a model polymer due to its extensive use in electrospinning48, combined with its biocompatibility and cost-effectiveness. Additionally, surface modification exploits the large surface area of EFs, and provides a useful alternative that can be combined with encapsulation to maximize fiber utility49. Unlike traditional encapsulation methods where only a portion of GRFT is available (and only transiently present in the FRT), surface modification may enable GRFT to maintain maximum bioactivity during the entire duration of treatment. Furthermore, the incorporation of hydrophilic compounds such as proteins, by traditional electrospinning methods, may result in lower encapsulation efficiencies and loss of protein activity50. Therefore, GRFT surface-modified fibers may offer a promising alternative delivery method that can be used alone or in combination with electrospinning to enhance protection against STI infection.

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fabrication and characterization of griffithsin-modified fiber scaffolds for prevention of sexually transmitted infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

Fiber morphology has a significant effect on the ability of surface-modified EFs to provide protection against viruses. Although electrospinning is a convenient and straightforward procedure, non-optimized polymer formulations may result in irregular fiber morphology (Figure 5B-C). Alterations in electrospinning conditions that result in the formation of beaded or amorphous mat-like morphologies, are often caused by solvent-polymer incompatib...

Watch this Scientific Journal Video about Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections at JoVE.com

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

The overall goal of this procedure is to fabricate and surface-modify polylactic acid, co-glycolic acid, electrospun fibers, with varying amounts of the antiviral protein Griffithsin. This method can help answer key questions in the drug delivery field, such as how to formulate and characterize new delivery vehicles that can prevent sexually transmitted viral infections, such as HIV-1. The main advantage of the technique is that the antiviral activity of Griffithsin is maximized by using polymeric-electrospun fibers as a stationary scaffold to highly localized Griffithsin on the fiber surface.The implications of this technique extend toward therapy of other viral and bacteria infections, because of Griffithsin's potent prophylactic and therapeutic potential against a variety of infection types. Though this method can provide insight into preventing HIV-1 infection, it can also be applied to other systems, such as other viral or bacterial infections in which Griffithsin has demonstrated potency. To begin this procedure, weigh 720 milligrams of 50/50 PLGA into a 10 milliliter scintillation vile.Using a serological glass pipette, add 3.0 milliliters of HFIP. Cover the vile with plastic film. Then, measure and record the vile mass.Incubate the polymer suspension at 37 degrees Celsius overnight to ensure complete dissolution of the polymer. After this, prepare the electrospinning apparatus as outlined in the text protocol. Use a three milliliter syringe to aspirate the polymer solution.Next, connect a blunt 18 gauge 0.5 inch needle tip to the syringe. Dispense the the excess solution to remove empty head space in the needle. Place the syringe on a syringe pump, and set the instrument flow rate to 2.0 milliliters per hour.Connect the power source to the syringe needle. Electrospin the polymer solution using a voltage of positive 27 kilovolts. After the entire solution is electrospun, turn off the power source.Allow the mandril to spin for an additional 30 minutes, to fully evaporate the solvent. Then, turn off the rotating mandril collector. Using a razor blade, gently peel the fiber from the mandril.Collect the electrospun PLGA fiber into a labeled petri dish. Place the dish in a desiccator overnight to remove any residual solvent. To begin, prepare the PBS and MES solutions as outlined in the text protocol.Next, remove the EDC and NHS from the freezer. Let them rest to equilibrate to room temperature. After this, weigh four milligrams of EDC into a 1.5 milliliter micro-centrifuge tube.Weigh six milligrams of NHS into a different micro-centrifuge tube. Add one milliliter of MES buffer to each tube. Vortex both tubes vigorously to ensure the reagents are fully dissolved.Then, weigh 70 milligrams of hydroxylamine into a 50 milliliter conical centrifuge tube. Add 20 milliliters of PBS, and vortex. Mass out an appropriate amount of PLGA fiber into a 15 milliliter conical centrifuge tube.Add eight milliliters of MES buffer to the fiber. Next, add one milliliter of both the EDC and NHS solutions. Use plastic film to seal the 15 milliliter conical centrifuge tube.Place the tube on a rotator to gently invert the solution for 15 minutes at room temperature. After this, add 14 microliters of beta-mercaptoethanol to quench the reaction. Invert the tube several times to ensure complete mixing.Discard the supernatant, and rinse the PLGA fiber twice using 10 milliliters of PBS for each wash. Add an appropriate amount of GRFT stock solution to the tube, as outlined in the text protocol. Then, add enough PBS to bring the final volume to eight milliliters.Close and invert the tube to ensure thorough mixing. Seal the tube with plastic film. Place the tube on the rotator to gently invert the solution for two hours.After this, quench the reaction by adding two milliliters of the prepared hydroxylamine solution, and vortexing for 30 seconds. Discard the supernatant. Rinse the surface modified PLGA fiber twice, using 10 milliliters ultra pure water, and vortexing for each wash.Next, transfer the washed fiber to a petri dish. Place the petri dish inside of a desiccator, until the fiber is completely dry. Then store the petri dish at four degrees Celsius.To begin, set out three 1.5 milliliter micro-centrifuge tubes. Mass out two milligrams of fiber into each. Then, add one milliliter of DMSO to each tube.Vortex for one minute at room temperature to completely dissolve the fiber. After this, dilute a 10 microliter aliquot at least 100 fold, in TE buffer. Store at minus 20 degrees Celsius, until loading characterization with Eliza.Weigh out between 5 and 10 milligrams of surface modified fiber, and transfer it to a micro-centrifuge tube. Record the fiber mass placed in each tube. Next, add one milliliter of a solution that mimics the physiological environment of each sample.Incubate the samples on a rotating shaker at 200 RPM and 37 degrees Celsius for one hour. After this, remove approximately one milliliter of the solution containing the desorbed GRFT and aliquot it into cluster tubes. Store at minus 20 degrees Celsius until ready to perform protein quantification.Then transfer the sample to a new micro-centrifuge tube. Add one milliliter of fresh buffer solution and incubate until the next time point, as outlined in the text protocol. In this study, the conditions for fabricating PLGA electrospun fibers are examined.SEM analysis of blank, 0.05 nanomole, 0.5 nanomole, and 5 nanomole GRFT fibers indicates that GRFT modification has no effect on fiber morphology. All of the formations are seen to have similar average diameters, around 1.9 microns, demonstrating the consistency of the modified fabrication process across batches. The amount of GRFT conjugated in the electrospun fibers is then determined.The 5 nanomole modification contains 373 nanograms of GRFT, while the 0.5 and 0.05 modifications contains 165 and 42, respectively. This demonstrates that fibers conjugated with higher theoretical surface modification density result in more GRFT conjugated to the fiber. However, an inverse correlation can be seen in the resulting conjugation efficiency.Next, the amount of GRFT covalently conjugated to the fiber is assessed. Within the first four hours, 113 nanograms, 25 nanograms, and 10 nanograms of GRFT per milligram of electrospun fiber is detected in the 5 nanomole, 0.5 nanomole, and 0.05 nanomole theoretical modification concentrations, respectively. After four hours, negligible GRFT is detected in the release elute for all three formulations.This data indicates that the majority of GRFT is covalently bound to the electrospun fibers and that the surface absorbed GRFT is released within the first four hours. Once mastered, this technique can be done in approximately four days. Electrospinning the polymer fiber scaffold, requires two days to prepare the polymer solution and to electrospin.Whereas surface modification, subsequent drying and fiber characterization required at least two additional days. While attempting this procedure, it's important to remember to maintain the proper ratios of reagents to ensure optimal modification. Furthermore, it's also important to verify that the electospinning parameters are correct to ensure appropriate fiber morphology.After its development, this technique paved the way for researchers in the field of drug delivery to fabricate electrospun fibers, that not only encapsulate antiviral agents within the fiber, but have a capacity to conjugate active agents through the fiber surface. After watching this video, you should have a good understanding of how to electrospin polymeric fiber, perform surface modification of fiber with the anti-viral protein Griffithsin, and qualitative Griffithsin conjugated to the fiber. Don't forget that working with reagents such as HFIP, beta-mercaptoethanol, EDC and NHS can be extremely hazardous.Precautions such as proper application of PPE, and the use of chemical fume hoods, should always be taken while performing this procedure.

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