Shell Nanofibers: In

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Tenofovir containing thiolated chitosan core/ shell nanofibers: in vitro and in vivo evaluations Jianing Meng, Vivek Agrahari, Miezan J. Ezoulin, Chi Zhang, Sudhaunshu S Purohit, Agostino Molteni, Daniel Dim, Nathan A. Oyler, and Bi Botti C. Youan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00739 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Molecular Pharmaceutics

Tenofovir containing thiolated chitosan core/shell nanofibers: in vitro and in vivo evaluations Jianing Meng a, Vivek Agrahari a, Miezan J. Ezoulin a, Chi Zhangb, Sudhaunshu S. Purohit b, Agostino Moltenic, Daniel Dimd, Nathan A. Oyler b, Bi-Botti C. Youana*

a

Laboratory of Future Nanomedicines and Theoretical Chronopharmaceutics Division of

Pharmaceutical Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64108, US b

Department of Chemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110,

US c

School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri 64108, US

d

Truman Medical Center Hospital Hill, Kansas City, Missouri 64110, US

Jianing Meng, Vivek Agrahari, and Miezan J. Ezoulin contributed equally

* Correspondence to: Bi-Botti C. Youan , Tel: (1)816-235-2410; Fax: (1)816-235-5779; Email: [email protected]

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ABSTRACT It is hypothesized that a thiolated chitosan (TCS) core/shell nanofiber (NF) can enhance the drug loading of tenofovir, a model low molecular weight and highly water soluble drug molecule, improve its mucoadhesivity, and in vivo safety.

To test this hypothesis, polyethylene oxide (PEO) core with TCS and polylactic acid (PLA) shell NFs are fabricated by a coaxial electrospinning technique. The morphology, drug loading, drug release profiles, cytotoxicity and mucoadhesion of the NFs are analysised

using

scanning

and

transmission

electron

microscopies,

liquid

chromatography, cytotoxicity assays on VK2/E6E7 and End1/E6E7 cell lines and Lactobacilli crispatus, and periodic acid colorimetric method, respectively. In vivo safety studies are performed in C57BL/6 mice followed by H&E and immunohistochemical (CD45) staining analysis of genital tract. The mean diameters of PEO, PEO/TCS, PEO/TCS-PLA NFs are 118.56, 9.95, and 99.53 nm. The NFs exhibit smooth surface with average diameters. The drug loading (13%25%, w/w) increased by 10 folds compared to a nanoparticle formulation due to the investigation of the electrospinning technique. The NFs are non-cytotoxic at the

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concentration of 1 mg/ml. The PEO/TCS-PLA core/shell NFs mostly exhibit a release kinetic following Weibull model (r=0.9914), indicating the drug release from a matrix system.The core/shell NFs are 40-60 fold more bioadhesive than the NFs of PEO only. The NFs are non-toxic and non-inflammatory in vivo after daily treatment for up to 7 days. Owing to their enhanced drug loading and preliminary safety profile, the TCS core/shell NFs are promising candidates for the topical delivery of HIV/AIDS microbicides such as tenofovir.

KEYWORDS: Nanofiber, coaxial electrospinning, mucoadhesion, drug loading, water soluble drug.

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List of abbreviation AAALAC

Association for Assessment and Accreditation of Laboratory Animal Care

BSA

body surface area

BZK

benzalkonium chloride

CS

chitosan

EC50

half maximal effective concentration

EDC

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

H&E

hemotoxylin and eosin

HPLC

high performance liquid chromatography

LARC

Laboratory Animal Resource Center

LDH

lactate dehydrogenase

IACUC

Institutional Animal Care and Use Committee

NF

nanofiber

NHS

N-hydroxysuccinimide

NP

nanoparticle

NRTI

nucleotide analogue reverse transcriptase inhibitor

PAS

periodic acid/Schiff

PBS

phosphate buffered saline

PEO

polyethylene oxide

PLA

polylactic acid

SEM

scanning electron microscopy

TCS

thiolated chitosan 4


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TFV

tenofovir

TEM

transmission electron microscopy

VFS

vaginal fluid simulant buffer

TPP

sodium triphosphate

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1. Introduction Tenofovir (TFV) is an anti-HIV topical microbicide belonging to the category of nucleotide analogue reverse transcriptase inhibitor (NRTI) proven to be effective in HIV/AIDS prevention and treatment1-3. To protect women from HIV transmission, development of a female controlled-prevention method is essential4. Therefore, vaginal delivery of TFV is promising in reducing of HIV-1 transmission in women5. Various polymer-based nanoparticles (NPs) and microparticles delivery systems have been developed for the vaginal delivery of TFV6-11. In these formulations, although the controlled release has been achieved, the low molecular weight and high water solubility of TFV lead to the loss of drug in the aqueous phase during the preparation process, leading to low encapsulation efficiency within the polymeric matrix. The study of nanomedicine has been dominated by spherical particle design, mainly because it minimizes the interfacial energy during the particles formation12. However, very few non-spherical delivery systems are studied even though they could potentially exhibit superior properties. One of the most promising non-spherical delivery systems are nanofibers (NFs), which can be designed by electrospinning13. Electrospinning utilizes electrostatic forces as its driving force to produce polymeric fibers14. It provides an effective and alternative way to directly encapsulate both hydrophobic and hydrophilic drugs into the fibers15. Biocompatible polymers can be electrospun into nano-sized mesh that is composed of fibers with a high surface-to-volume ratio, which is desirable for mucoadhesive drug delivery systems16,

17

. Moreover, the strong capability of multiple-fluid

electrospinning processes (such as coaxial, tri-axial and side-by-side) in tailoring material compositions within the NFs structure makes it possible to create surface with improved mucoadhesive property 18-20.

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Thiolated chitosan (TCS) is used as the mucoadhesive material in NF formulation. Its mucoadhesive property has been proven in various studies21, 22. TCS on the surface of the formulation can bind to the mucosa, which lines the surfaces of body cavities, such as the vagina and rectum, to maintain a certain level of moisture23. Therefore, the adhesive property of a formulation is largely depended on its surface-to-volume ratio. Compare to spherical NPs, NFs have higher surface-to-volume ratio resulting in more interfacial area available to interactions thereby aiding the adhesion24, 25. Once applied topically, the mucoadhesive NFs intend to stay in close contact with the mucosa, prolong the residence time on the application site, provides long-lasting protection to women26, since heterosexual contact is the major route of acquiring HIV for women27. In this study, it is hypothesized that a TCS core/shell nanofiber (NF) can enhance the drug loading of TFV, a model low molecular weight and highly water soluble drug molecule, improve its mucoadhesivity, and in vivo safety. 2. Materials and methods 2. 1. Materials Chitosan (CS) (Mw 50-190 kDa), thioglycolic acid, poly(ethylene oxide) (PEO) (Mw 900 kDa), formic acid, mucin (Type III), periodic acid Schiff reagent kit and DAB: Peroxidase Substrate Kit are purchased from Sigma Aldrich (St. Louis, MO, USA). Poly (DL-lactide acid) (PLA) (PDL 05) is a gift from Purac Biomaterials (Lincoln, IL, USA). 1ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride

(EDC)

and

N-

hydroxysuccinimide (NHS) and sodium triphosphate penta-basic (TPP) are supplied by supplied by Thermo Fisher Scientific (Pittsburgh, PA, USA). TCS is synthesized as described in the Supplementary material file. Tenofovir (TFV) is purchased from Zhongshuo Pharmaceutical Co. Ltd. (Beijing, China). The CytoTox-ONE™ and CellTiter 7



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96™ Aqueous kits are purchased from Promega (Madison, WI, USA). Anti-CD45 and biotinylated secondary antibody is obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). 2.2. Experimental section 2.2.1. Preparation of the core/shell NFs A blend of TFV and PEO is used as the core material. A concentric spinneret is used to allow for the injection of PEO solution into the TCS solution as shell material at the syringe tip. The core composite solution is typically prepared by dissolving PEO into 50% v/v formic acid aqueous solution and stirring over night to yield a homogeneous solution. Then TFV is dissolved in the same solution. To prepare the shell composite solution, TCS or TCS-PLA blend is dissolved in pure formic acid at the concentration of 3% (w/v) and TPP is added in the solution at the ratio of TCS:TPP is 8:1 (w/w) (Table 1). An electrospinning system with a coaxial nozzle (NaBond Technologies Co., Ltd., HongKong, China) is used for NF production. The core and shell composite solutions are placed in two separate 30cc BD Multifit™ glass syringe (Becton, Dickinson and Company, NJ, USA). Two Cole-Parmer 78-0100C Digital Programmable Syringe Dispensing Pumps (Cole-Parmer Instrument Company, IL, USA) are used to supply a continuous and constant amount of solution. The feeding rate of both the solutions is 50 µl/h. A voltage of 15 kV is applied to the nozzle by a Gamma ES30 high voltage power supply (Gamma High Voltage Research, Inc., FL, USA). The NFs are collected onto an aluminum collector connected to the ground. The distance between the collector and the syringe tip is 10 cm. After electrospinning, the prepared NF mat is peeled from the surface of the collector and stored in a vacuum desiccator. 2.2.2. Characterization of the NFs 8


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2.2.2.1. Percent drug loading and yield determination To determine the drug loading, approximately 1 mg of TFV loaded NFs is submerged in 50 ml water for 48 h to allow complete dissolution of the PEO core and vortexed for 5 min. After centrifugation (Micro 18R Ultracentrifuge, VWR International LLC., PA, USA) at 10,000 rpm for 20 min, the supernatant is analyzed by using a high performance liquid chromatography (HPLC) (Waters, Milford, MA) assay at 259 nm28. The percent drug loading is calculated by using Equ. 1: Drug loading % =

Total amount of TFV × 100% （1） Total amount of NFs

The yield of the NFs fabrication process is calculated by the ratio of total mass of the electrospun NFs to the total mass of the initial ingredients combined (Equation 2). Yield % =

Total mass of NF × 100% （2） Total mass of initial components combined

2.2.2.2. Morphological and surface chemical analysis The morphology and surface characteristics of the NFs are analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In order to elucidate the surface chemistry, the NFs are directly scanned over the wavenumber range of 650 and 4000 cm−1 using a Nicolet™ iS™10 FT-IR Spectrometer (Thermo Scientific, Lenexa, KS, USA). (See supplementary online material for further details.) 2.2.3. In vitro drug release profile from the NFs One milligram of NFs is immersed in a tube containing 40 ml of vaginal fluid simulant buffer (VFS), prepared according to previous reports29. The tubes are kept in a thermostatically controlled water bath (BS-06, Lab Companion, USA) at 60 rpm, the temperature is maintained at 37ºC. At predetermined time intervals, 1 ml of the VFS buffer solution is taken out and replaced by fresh buffer to maintain the sink conditions. 9



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The concentration of drug in the buffer solution is determined by the above HPLC assay. Each experiment is run in triplicate (n = 3) together with a blank control30. The release profiles of the PEO/TCS and PEO/TCS-PLA core/shell NFs are simulated to a series of known release kinetic models: first-order model, Higuchi model, Korsmeyer-Peppas model, and Weibull model31, The model with the highest determination coefficient (r2) is chosen as the best fit, data is analyzed using the Excel add-in DDSolver program32. In order to compare release kinetics of the PEO/TCS and PEO/TCS-PLA core/shell NFs with the non-core/shell PEO NFs, a simple model independent approach of FDA guidance is applied. The difference factor (f1) and similarity factor (f2) are calculated

33

.

f1 is a measurement of the percent difference between the reference and test sample at each time point, while f2 calculates the similarity in the percent dissolution between the two samples. Two curves can be considered similar if the values of f1 and f2 are close to 0 and 100, respectively. 2.2.4. Mucoadhesion study This study works respectively with the following two methods: vaginal tissue infusion method, a qualitative biological method; and periodic acid/Schiff (PAS) colorimetric method, a quantitative non-biological method. Firstly, to assure that the NFs can adhere to the vaginal mucus, the porcine vaginal tissues are obtained from the local abattoir (Fairview Farm Meat Co., Topeka, KS, USA). These tissues are washed using normal saline, snap frozen in liquid nitrogen, and then stored at -80°C. Prior to the experiment, the frozen tissues are thawed in normal saline at 37°C for 3 min. The fluorescein isothiocyanate (FITC) labeled TCS is then synthesized as previously described34. The tissue is cut into pieces of 2 cm length and 1 cm width and stuck to a plastic strip by cyanoacrylate glue, which is resistant to water and harmless to the tissue. The average 10


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weight of 100 cm2 NFs mesh is 10 mg. Therefore, 1 cm2 (equivalent to 0.1 mg) of FITC labeled

PEO/TCS and PEO/TCS-PLA NFs are distributed over the surface of the tissue. The strip is put into a tube that is placed in a water bath of at an angle of 75° to allow buffer flow by gravity. The tissue is then placed in a container maintains at 37°C and relative humidity for 20 min to allow the NFs to hydrate and to interact with the mucin layer of the tissue. After 20 min, the mucosa is continuously washed for 10 min with VFS at the rate of 1 ml/min. The tissue is then cut into small cubes, embedded into Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA), frozen by Histological Freezing Spray (Cardinal Health Inc., Dublin, OH, USA), and sectioned vertically using a Leica CM1950 cryostat microtome (Leica Biosystems Inc., Buffalo Grove, IL, USA). The skin sections (6 mm) are mounted on glass slides and visualized through a fluorescence microscopy (Nikon, Shizuoka Prefecture, Japan)35. Secondly, to assess the amount of mucin adsorbed on the NFs, periodic acid reagent is freshly prepared by adding 10 µl of 50% periodic acid solution to 7 ml of 7% (v/v) acetic acid solution. One milligram of PEO, PEO/TCS, and PEO/TCS-PLA NFs is added in the mucin solution of VFS at the concentration of 1 mg/ml. The solution is incubated in a shaking water bath for 30 min at 37°C to enable NFs-mucin interaction. Then, the dispersions are centrifuged at 4000 rpm for 2 minutes to separate NF-mucin conjugate, and the supernatant is used for the measurement of the free mucin content. For each sample, 0.2 ml of periodic acid reagent is added, the samples are incubated at 37°C for 2 hours in a water bath. Then, 0.2 ml of Schiff reagent is added at room temperature. Thirty minutes later, the absorbance of the solution is recorded at 555 nm by using the Genesys 10 Bio UV–Vis Spectrophotometer (Thermo Electron Sci. Inst., LLC, Madison, WI, USA). Standard calibration curve is prepared from 1 ml of mucin standard solutions 11



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in the concentration range of 0.25 - 2 mg/ml36. The mucin content is calculated from the standard calibration curve. The mucin solution without NFs is used as a control and assessed using the above procedure. Each experiment is performed in triplicate (n = 3). The percentage of mucin adsorption is estimated using Equation 3: Mucin adsorption % =

Total mass of mucinmated using × 100% （3） Total mass of mucin

2.2.5. In vitro cytotoxicity studies Human vaginal keratinocyte cell line (VK2/E6E7) and human endocervical epithelial cell line (End1/E6E7) are grown in the Keratinocytes-SFM medium, the media is supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells are cultured at 37°C in a humidified 5% CO2 atmosphere and sub-cultured prior to confluence using trypsin. Lactobacilli crispatus is grown in an ATCC medium 416 Lactobacilli MRS broth (BD, Franklin Lakes, NJ, USA) and cultured at 37°C in a humidified 5% CO2 atmosphere. Cytotoxicity of the NFs is determined using cellular membrane integrity assay, cell viability assay, and Lactobacillus viability assay (See supplementary online material for further details)30. 2.2.6. In vivo safety studies in C57Bl/6 mice The animal experiments are duly approved by the University of Missouri - Kansas City (UMKC) Institutional Animal Care and Use Committee (IACUC). Female 8-12 weeks old C57Bl/6 mice, weighing about 18-22 g, are obtained from Jackson Laboratories (Bar Harbor, Maine, USA.) and allowed to acclimate for 7 days. All mice are housed in the UMKC Laboratory Animal Resource Center (LARC) facility which is a fully Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited. In order to maintain a constant diestrus-like vaginal cytology for reproducible experimental

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conditions, mice are s.c. injected with 2 mg of medroxyprogesterone acetate (DepoProvera®, Greenstone, Peapack, NJ, USA)) in a total volume of 200 µl of in Lactated Ringer’s saline solution 4-5 days before the treatment to induces and maintain a diestrus-like state.. The mice are divided randomly in three groups (n = 4). Group 1 is treated with 20 µl Benzalkonium chloride (BZK, 2 %w/v), as positive control. Group 2 is treated with phosphate buffered saline (PBS) is used as negative control; Group 3 is treated with freshly prepared PEO/TCS-PLA NFs. The NFs equivalent drug dose of 475 mg/kg is administrated to each treated mouse. The intra-vaginal instillation is carried out once daily using a flexible feeding needle (Cadence, Inc. Staunton, VA, USA). After 1-day and 7-day intravaginal application, mice are euthanized by carbon dioxide (CO2) asphyxiation. Their vaginal tissues are collected, formalin fixed in paraffin, and cut into 5-mm sections. Serial sections are stained with hemotoxylin and eosin (H&E). In order to identify the inflammatory cell (CD45) infiltrate, immunohistochemical staining is performed in genital tissue sections (See supplementary online material for further details)37. 2.2.7. Statistical data analysis The results are expressed as the mean of at least 3 experiments (n=3) ± the standard error of the mean. Statistical data analysis is performed using the student t-test; two tails with 95% confident interval (p < 0.05) as the minimal level of significance. 3. Results 3.1 Characterization of the NFs 3.1.1. NF drug loading and percent yield As shown in Table 1, the drug loading of the PEO, PEO/TCS and PEO/TCS-PLA NFs, 13



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are in close agreement with the theoretical values indicating that 100% of drug is encapsulated. Their respective yields are 102.48%, 53.65% and 63.18%. The jet stretches by whipping and bending. The nature of the whipping motion and bending instability of the charged jet in the electrospinning process may cause some shift in the direction, and distribute the fibers over a larger area. For PEO/TCS and PEO/TCS-PLA NFs, the area of NFs deposit exceeds that of the collector, not all the NFs can be collected on the aluminum foil, which lead to a lower yield (Table 1). 3.1.2. Morphology studies The SEM images (Figure 1) shows that the pure PEO fibers and the core-shell fibers have been successfully produced using the electrospining technique. To characterize the NFs, the average fiber diameter, average bead diameter, and the approximate ratio of the bead area to the total NFs area are summarized in Table 1. Both PEO and PEO/TCS-PLA NFs are highly smooth without the occurrence of beads. However, for the PEO/TCS NFs, beads with the mean diameter of 58.81 nm are observed.

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Fig. 1 15



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To further identify the core/shell structures, the morphological detail of the produced core/shell NFs is shown in a TEM micrograph in Figure 1 D-G. In Figure 1 D and E, the core appears darker than the shell. The electrospun NFs are then soaked in water in order to selectively remove the PEO, since the water is a good solvent for PEO but poor solvent for TCS and TCS-PLA blend. The structure of the resulted NFs are showed in Figure 1 F and G. It appears that the PEO/TCS NFs are broken after aqueous extraction; fragments of the NFs instead of intact fibers are observed, revealing a discontinuous core–shell structure. Comparatively in Figure 1 G, after the core (darker region) is completely removed, an integrated tube-like structure is observed. The PEO/TCS-PLA NFs show very uniform core–shell structure with obvious boundary: the core measures about 30 nm while the shell is approximately 20 nm in thickness. According to the SEM and TEM image, it is speculated that the PEO/TCS NFs have a necklace-like structure (Figure 1 H), where TCS beads of different size are on the PEO string. Unlike PEO/TCS NFs that have a fine core/shell structure (Figure 1 I), PEO is mainly located in the core while TCS-PLA blend constitutes the shell. 3.1.3. FTIR analysis Figure 2 displays the FTIR spectra of native PEO, TCS, PLA and the core/shell NFs. The native PEO shows a characteristic band at 2890 cm−1 corresponding to the CH2 stretching vibration, which overlaps with that of chitosan. Absorption bands of hydroxyl (OH) groups are negligible as a high molecular weight (900 kDa) PEO is used. Other feature bands of PEO observed from 1240 to 1360 cm–1 are due to CH deformation of the methyl groups (-CH3). The triplet peaks at 1065, 1095 and 1150 are referred to C−O−C stretching vibrations38. The native TCS exhibited a broad absorption peak at around 3100 to 3650cm–1 and weak absorption peaks at 2890 cm–1, which are attributed 16


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to N-H and OH-O (hydrogen bond) stretching vibration and the CH stretch, respectively. The two peaks at 1520 and 1650 cm–1 are respectively due to the amide (CO-NH-) and the amine (-NH2) absorption band. The FTIR spectra of native PLA reveals a sharp peak at 1750 cm−1 and characteristic bands from 1050 to 1200 cm−1, 1187, 1131 and 1093 are assigned to the ester groups (-CO-O-) of PLA39 . The peak of 1758 cm−1 which belonged to the carbonyl group in PLA is decreased.

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Fig 2 18


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In the spectrum of the PEO/TCS NFs, the absorbance intensity of amide and amine groups stretching at 1520 and 1650 cm-1 is decreased and the absorbance intensity of CH2 stretching vibration at 2890 cm-1 is increased, indicating that the PEO exists not only in the inner core, but also on the surface of the NFs. For the PEO/TCS-PLA NFs, the increasing of the peak at 2890 cm-1 is not apparent, three characteristic bands at 1520, 1650 and 1750 cm-1 are observed, suggesting that the shell of these NFs are composed mainly of TCS and PLA, no PEO or perhaps a very minute quantity of PEO distributes on the surface of the NFs due to the fine core–shell morphology. 3.2. In vitro drug release study The drug release profiles for the NFs are shown in Figure 3. For the PEO NFs, approximately 95% of drug is released within 5 h, which is attributed to the hydrophilic nature of PEO. The mechanism of drug release is based on dissolution/diffusion of the drug. The result model fitting of the drug release kinetic from the core/shell NFs is summarized in Table 2. The first order model and Higuchi model represent the release kinetics controlled by concentration and diffusion respectively. The n value calculated from the Krosmeyer-Peppas model dependent on the extent of cross-linking. the n values are in the range of 0.50 to 1.0, indicating that the drug release from the NFs follows nonFickian trends

40

. The best fit of both PEO/TCS and PEO/TCS-PLA NFs is observed with

the Weibull model, suggesting that the drug is released from a matrix type delivery system. The matrix is due to the ionic cross-linking between TPP and TCS that forms a three-dimensional network 41. Given the fact that the effective molar half maximal effective concentration (EC50) of TFV is 5.0 to 7.6 µM, if the concentration of the NFs in VFS is 1 mg/ml, it is equivalent to a TFV concentration of 350 µM based on the average drug loading (~10%). This means 19



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that the NFs could exhibit its anti-viral activity when approximately 2.1% of drug is released from the NFs in the release media/. According to the predicted release profiles in Figure 3, both PEO/TCS and PEO/TCS-PLA NFs could release 2.1% of TFV within 2 min after contacting with release media. Therefore, it is reasonably speculated that both NFs are able to provide sufficient TFV concentration to exhibit an anti-HIV activity.

Fig. 3 For both PEO/TCS and PEO/TCS-PLA NFs, their f1 values are higher than 0; f2 values are lower than 100, indicating significant difference between the PEO NFs and the core/shell NFs. After 24h, more than 80% of the drug is released from the PEO/TCS NFs, whereas approximately 60% of the drug is released form the PEO/TCS-PLA NFs. The release rate of the former is faster due to its discontinuous core–shell structure. Overall, the PEO/TCS-PLA NFs provide a more sustained control on the drug release. 3.3. Mucoadhesive studies The mucoadhesion of PEO/TCS and PEO/TCS-PLA NFs are assessed following the infusion procedure. As illustrated in Figure 4 A, after rinsing using VFS, the fluorescence intensity of both NFs is not markedly reduced. Both NFs are

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spontaneously adsorbed to the surface of the porcine vaginal tissue, as a strong interaction exists between TCS and mucin. Moreover, the interconnected NFs inseparably associates with each other, and thus constituting a network, even if part of the NFs is detached from the tissue the integrated network would not peel off. These results confirm that both PEO/TCS and PEO/TCS-PLA NFs are resistant to vaginal fluid and subsequently might increase the retention time in vivo.

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Fig. 4 22


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The amount of mucin adsorbed on the NFs is shown in Figure 4 B, low (0.2 mg /ml), medium (1 mg/ml), and high (2 mg/ml) concentration of mucin is selected, 45%-65% mucin is trapped by PEO/TCS-PLA NFs and PEO/TCS NFs with differed mucin concentration, as a comparison, less than 7% mucin is adsorbed on PEO NFs. These results confirm that both PEO/TCS and PEO/TCS-PLA NFs have the ability to adsorb mucin and potentially enhance mucoadhesion in vivo. 3.4. In vitro cytotoxicity assays A vaginal drug delivery system must have safety profiles that justify its application,

especially for the vaginal ecology42. Thus, it is important to test the impact of the drug-loaded NFs on the viability and structural integrity of the vaginal epithelial cells as well as the natural flora in the vagina. The cellular membrane integrity is determined by the release of lactate dehydrogenase (LDH) from cells with damaged membranes. MTS is a tetrazolium compound that is convertible into a brown colored formazan product by viable cells with active metabolism, the amount of formazan produced is proportional with the number of viable cells43. Figure 5 A-D shows the results of the LDH release and the cellular viability (MTS assay) using the selected vaginal epithelial (VK2/E6E7) cell line and human endocervical epithelial (End2/E6E7) cell lines. After 24 h or 48 h, the TFV loaded NFs cause a statistically non-significant (p>0.05) release of LDH as a comparison with that caused by the media. The cellular viability test results are consistent with those of LDH assay, as no statistical difference (p>0.05) is observed by the Student t-test between the media and the TFV loaded NFs. The lactobacillus viability assay (Figure 5 E-F) also shows that no statistically significant loss of bacterial viability is observed during the incubation period. The results indicate a

23



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non-cytotoxic nature of the NFs on both vaginal and endocervical epithelial cell lines and the vaginal flora at the concentration of 1 mg/ml up to 48 h.

Fig. 5 3.5. In vivo safety studies in C57Bl/6 mice In order to determine whether the exposure of the MPs results in inflammation and toxicity to the vaginal mucosa, the H&E stained vaginal sections of the mouse are evaluated. The PEO/TCS-PLA NFs contain the equivalent drug dose of approximately 65 mg/kg TFV, which is 8-fold higher than the effective dose (8 mg/kg), after convertion 24


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from the effective human dose (40 mg/person)2 base on body surface area (BSA) normalization method44. Optical microscopy examination of H&E staining indicates intact vaginal epithelium and the lack of leukocyte influx in the representative vaginal sections from mouse treated with NFs daily for 1 day (Figure 6 top row), and 7 days (Figure 6 bottom row). These observations consistent with the pathological data of tissue from mice treated with PBS (negative control). In contrast, vaginal tissue sections following daily intravaginal administration of with 2% BZK (positive control), reveals extensive denudation of the epithelial cell layer and leukocyte infiltration (red arrows in Figure 6)45.

Fig 6 The CD45-associated protein is a lymphocyte-specific membrane protein. The increasing of the lymphocytes infiltration within the vagina epithelium is an indication of the vaginal inflammation37. After NFs application intravaginally, the distribution of CD45 positive cells in the vaginal tissue and the integrity of the vaginal epithelium is similar to that of PBS-treated (negative control) tissues. However, the number of the CD45positive cells is elevated within the tissue treated by the 2% BZK (positive control) as the

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red arrows shown in Figure 7. These observations suggests that no infiltration of immune cells is induced due to the MPs treatment.

Fig 7 4. Discussions Electrospinning is a simple, flexible, and cost-effective process that can form a broad range of polymeric fibers. During the electrospinning process, when a sufficiently high electrical potential is applied to the droplet of polymer solution, the droplet becomes charged. At a critical voltage, the electrical energy overcomes the surface energy of the polymer droplet, and a jet erupts from the surface. The jet is then elongated under the electrostatic force while the solvent of the polymer is being evaporated until it is finally deposited on a grounded collector46-48. TCS is a derivative of chitosan, it presents on the surface as the shell can interact with the mucosa while the TFV is released from the core in controlled manner. Chitosan is a cationic polysaccharide. The amino groups on the polymer backbone are ionizable and positively charged under acidic condition. During the electrospinning process, the

26


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repulsive force between ionic amino groups within polymer backbone limit its electrospinnability and inhibits the formation of continuous fiber under the high electric field49. Therefore, the electrospinning of pure TCS typically forms beads and droplets instead of ultrafine fibers50. To overcome this drawback and assure continuous nanofiber formation, the following three complimentary strategies are used. Firstly, TCS is blended with PLA, which is biodegradable aliphatic polyester, and has been approved by the FDA for human clinical applications. PLA is a good fiber-forming polymer as it exhibits excellent film forming mechanical properties, attractive molding and shaping properties51, 52. It can be fabricated into porous NFs and many other types of structures53,

54

. Moreover, PLA can help crosslink and solidify the shell by forming

hydrogen bound with chitosan55. Some works have been devoted to manufacture PLA/chitosan composites39, 56. In our preliminary study, it is observed that the obtained NFs became more uniform when equal amounts of TCS and PLA are used. Secondly, formic acid is the solvent of choice. This could be attributed to several reasons. 1) Formic acid is a good solvent of PLA/TCS composite, both TCS and PLA are soluble in formic acid. 2) The TPP is used to cross-link chitosan via charge-charge interaction between the phosphate groups of TPP and the amino groups of chitosan57. The crossing linking is dependent on the availability of the cationic sites and the anionic sites. Chitosan is polycationic in acidic environment, TPP has 5 pKa values as follow58, 59

: +

-

pK1 = -∞ (H5P3O10 ↔ H + H4P3O10 ) -

+

(4)

2-

pK2 = 1.1 (H4P3O10 ↔ H + H3P3O10 ) 2-

+

(5)

3-

pK3 = 2.3 (H3P3O10 ↔ H + H2P3O10 )

(6)

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3-

+

Page 28 of 44

4-

pK4 = 6.3 (H2P3O10 ↔ H + HP3O10 ) 4-

+

(7)

5-

pK5 = 8.9 (HP3O10 ↔ H + P3O10 )

(8) -

Since the pH of pure formic acid is lower than 1, only H4P3O10 ions are present, the negative charge is too low to cross-link the positively charged chitosan. TPP and chitosan are free in the solution. After electrospinning, when the NFs are immersed in the VFS (pH 4.2), both chitosan and TPP dissociate to provide sufficient cationic sites and the negative sites, not until then will they crosslink and form the insoluble shell. This is supported by the fact that the solution of chitosan and TPP in pure formic acid is dropped into water or VFS, it precipitates as white flocky sediment (Figure 8). 3) The vapor pressures of formic acid and acetic acid is 45 mm Hg and 11 mm Hg at 20 °C, respectively60. The relative higher volatility of formic acid is also advantageous for the rapid solidification of the electrified jet. 4) chitosan solution has high viscosity, which limits its spin ability61. As a comparison with other solvent of chitosan, such as acetic acid (intrinsic viscosity [η] = 28.21×102 dL/g), the viscosity of the solution of chitosan in formic acid (intrinsic viscosity [η] = 6.72×102 dL/g) is relatively lower at equal concentration62. During the electrospinning process, the polarity of the fluid that is generated by the voltage works against the surface tension of the solution to deform the droplet and form the Taylor cone. When the voltage (V) attains a critical value (VC), a jet is ejected from the cone and the fiber is formed. VC can be calculated as follows63: #

%$(% 2) = 4 ' % * +,− 1.52 0.1174.5 9 ) .

Where H denotes the gap distance, L is the length of the capillary tube, R is the radius of the tube, γ is the surface tension of the fluid. In this study, H = 10 cm, L = 2 cm,

28


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R = 0.6 mm.

Fig 8 The relationship between the liquid surface tension and its viscosity can be expressed using Equation 10: 57

8: 9

8

= ;< =>

(10)

Where A and B are characteristic constants of the liquid64. Therefore, the lower viscosity facilitates the formation of the jet by decreasing the surface tension. But very low viscosity leads to the loss of molecular entanglement in

29



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the solution, and makes difficult the formation of continuous fibers65. Hence, the viscosity of the fluid should be in a optimal range for effective NFs formation. Thirdly, PEO serves not only as a core material, but also a spinning aid. The PEO core solution is believed to act as a carrier that form a stable Taylor cone and draws out the shell chitosan solution by forming a continuous jet ejection66. Moreover, PEO is nontoxic and has been approved by the FDA for use in different pharmaceutical formulations67. To successfully prepare the core/shell NFs, 50% formic acid is used as the solvent of PEO. Using the same solvent in the two streams can reduce the interfacial tension between the two solutions. The surface tension of 50% formic acid at 20 °C is 43 dyn/cm, which is relatively close to that of the pure formic acid (37 dyn/cm at 20°C)68. This favors the coaxial electrospinning process69. If water (72 dyn/cm at 20°C) is used as the core solvent instead of the formic acid solution, it appears that no fiber can be formed due to inadequate viscosity and surfacetension. This result is also consistent with Pakravan et al study66. . Besides the morphological difference, there is a difference in diameter between the pure PEO NFs and the PEO/TCS ones. Compared to the pure NFs, the diameters of the PEO/TCS core shell NFs are significantly lower due to the strength of the repulsive force as explained above. The incorporation of PLA into the TCS shell solution leads to the formation of continuous, uniform, beads free, cylindrically-shaped fibers. In TEM, a beam of electrons is transmitted through the sample. The contrast, which represents the distinctive phases in the NFs, is due to the differential absorption of electron beam in the material with different density and thickness used in TEM samples70. To form an image, the final beam density I can be expressed as BC

? = ?@ A 7 D EFGH 11 30


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Where I0 is the intensity of the incident beam, NA is the Avogadro’s number, A is the atomic weight, σt is the total scattering cross-section of an atom, ρ is the density of the material, x is the thickness of the sample71. The decrease in transmission can be caused by the density and local thickness in the same way. The density of chitosan is relatively lower (approximately 0.3 g/cm3) than that of the PEO (1.21 g/cm3), Therefore in Figure 1 D to G, the PEO core appears darker than the shell owing to the high density of PEO. After aqueous extraction, PEO/TCSPLA NFs form a tube-like structure, the middle of the hollow tube is thinker than the edge, therefore the core is brighter than the shell. The enhanced mucoadhesive property of the core/shell NFs is based on the covalently attaching of thio-glycolic acid thiolation to chitosan backbone. TCS displays thiol side chains that mimic the natural structure of the secreted mucus glycoproteins. These side chains can form disulfide bound with the cysteine-rich subdomains of mucin via thiol/disulfide exchange reactions or a simple oxidation process of free thiol groups. As a result, TCS can covalently anchor in the mucus layer72, 73. The PAS technique is a widely employed colorimetric assay for the demonstration of mucin. The periodic acid solution is used as the oxidant. The periodic acid oxidizes the hydroxyl (OH) groups in the monosaccharide units of mucin, thus generating Schiff reactive aldehyde groups. These free aldehyde groups then react with the Schiff reagent to give a bright red magenta color. In this study, low (0.2 mg /ml), medium (1 mg/ml), and high (2 mg/ml) concentration of mucin is selected, due to the fact that the amount of mucin changes in the female reproductive tract during the menstrual cycle, the daily production of cervix mucus in a healthy woman is around 20–60 mg, which increases up to 700 mg/day and becomes less viscous. During the midcycle, the mucus compositional differences also 31



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exist among individuals74. The proportion of mucin adsorption of PEO/TCS-PLA NFs is similar to that of PEO/TCS NFs. The possible reason for this observation is that the thiolated groups exposed at the surface of the PEO/TCS-PLA NFs are no less than that exposed at the surface of the PEO/TCS NFs, since TCS forms discontinuous, large, or small beads while TCS-PLA blend forms continuous and uniform fiber shell. In contrast, little mucin is adsorbed on PEO NFs. 5. Conclusion In this study, the novel TFV loaded electrospun nanofibers are developed consisting PEO, TCS and PLA to enhance drug encapsulation efficiency and topical mucoadhesion. The engineered NFs exhibit a core-shell structure, with 13% to 25% w/w drug loading, sustained drug release profile, and enhanced mucoadhesion. Moreover, they are non-cytotoxic in vitro and in vivo, This is a significant improvement over previous reports, where various TFV loaded nanomedicines were prepared with low encapsulation efficiency and drug loading due to the high hydrophilicity of TFV. Due to these promising properties, TFV loaded NF can be considered as a good candidate for the delivery of water-soluble small-molecule drugs, and a promising vaginal delivery system for the prevention of HIV transmission. ACKNOWLEDGMENTS The work presented is supported by Grant Number R01A1087304 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institute of Allergy and Infectious Diseases or the National Institute of Health. We are grateful to Dr. Vladimir M. Dusevich (School of Dentistry, University of Missouri-Kansas City, MO) for the SEM and TEM.

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Table 1. Composition, drug loading, yield and mean diameter of the NFs. Data are given as mean ± SEM for n=3. NF

PEO NF

Concentration of each Component (%, w/v) Core solution Shell solution PEO 3 TFV 1 PEO 3 TFV 1 PEO 3 TFV 1

N/A

Drug loading (%)

Yield (%)

25.62±0.93

102.48±0.95

Mean diameter of fibers (nm) 118.56±4.77

TCS 3 13.42±3.51 53.65±0.26 9.95±0.64 TPP 0.38 TCS 1.5 14.20±2.12 63.18±1.16 99.52±5.52 TPP 0.19 PLA 1.5 Note: NF represents nanofiber, PEO represents, TFV represents, TCS represents, TPP represents, PLA PEO/TCS NF PEO/TCSPLA NF

represents. The solvent (10 ml) of the core and shell components is 50% formic acid and pure formic acid, respectively.

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Table 2. Estimated Parameters of the

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in vitro drug release kinetics and model independent analysis of

PEO/TCS and PEO/TCS-PLA core/shell NFs.

NFs

PEO /TCS PEO /TCS -PLA

Firstorder release model 2 k1 r 0.0 0.9 84 781 0.0 0.9 44 852

Higuchi release model kH 15.1 94 12.1 33

2

r 0.89 51 0.90 78

KrosmeyerPeppas release model kKP 14. 119 6.5 67

n 0.5 23 0.6 86

2

r 0.89 62 0.95 20

Weibull release model

α 17.3 5 37.1 4

β 1.17 9 1.17 5

2

r 0.98 30 0.99 14

Model independent analysis f1 41.23

f2 8.24

56.73

1.31

Note: k1, kH, kKP , andαβrepresent the release rate constant of different model, n is the release exponent, r2 represent the determination coefficient, f1 and f2 denote the difference factor and similarity factor, respectively.

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Figure Captions

Figure 1. Morphology of the nanofibers (NFs). Scanning electron microscopy (SEM) image of PEO NFs (A), PEO/TCS NFs (B), PEO/TCS-PLA NFs (C), Transmission electron microscopy (TEM) image of PEO/TCS NFs before PEO core extraction (D), PEO/TCS-PLA NFs before PEO core extraction (E), PEO/TCS NFs after PEO core extraction (F), PEO/TCS-PLA NFs after extraction (G) (Scale bar = 2 µm for SEM, scale bar = 200 nm for TEM), and schematic description of PEO/TCS nanofibers (NFs) (H) and PEO/TCS-PLA NFs (I).

Figure 2. The FT-IR reflectance spectra of neat PEO, TCS, PLA powder, PEO/TCS and PEO/TCS-PLA nanofibers (NFs). Figure 3. In vitro drug release profiles from PEO nanofibers (NFs), PEO/TCS NFs, and PEO/TCS-PLA NFs in vaginal fluid simulant buffer (n = 3). Dash lines represent the predicted release profiles following Weibull model. Data are shown as mean ± SEM (n = 3). Figure 4. Mucoadhesion of the nanofibers (NFs). A: fluorescence microcopy of thin sections of porcine vaginal tissue treated with PEO/TCS NFs, PEO/TCS-PLA NFs, and VFS without NFs before (top row) and after (bottom row) rinsing by vaginal fluid simulant buffer (Scale bar = 0.1 mm); B: adsorption of mucin on PEO, PEO/TCS and PEO/TCS-PLA NFs. Figure 5. Cytotoxicity study of PEO nanofibers (NFs), PEO/TCS NFs, and PEO/TCS-PLA NFs. CS and CS-TGA NPs. LDH release of VK2/E6E7 cell line (A) and End1/E6E7 cell line (B), percent viability of VK2/E6E7 cell line (C) and End1/E6E7 cell line (D), percent viability of L.cripatus after incubated with the NFs for 24 h (E) and 48 h (F). Data are shown as mean ± SEM (n = 3). *: p

Shell Nanofibers: In

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