Author's Accepted Manuscript Anatomic targeting improves delivery of unconjugated nanoparticles to the testicle Devon C. Snow-Lisy , Edmund S. Sabanegh , Jr., Mary K. Samplaski , Vinod Labhasetwar

PII: DOI: Reference:

S0022-5347(15)03373-X 10.1016/j.juro.2015.03.076 JURO 12465

To appear in: The Journal of Urology Accepted Date: 12 March 2015 Please cite this article as: Snow-Lisy DC, Sabanegh ES Jr., Samplaski MK, Labhasetwar V, Anatomic targeting improves delivery of unconjugated nanoparticles to the testicle, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.03.076. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

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Title: Anatomic targeting improves delivery of unconjugated nanoparticles to the testicle Authors: Devon C. Snow-Lisy M.D.a,1, Edmund S. Sabanegh, Jr. M.D.a, Mary K. Samplaski M.D.a,2, Vinod Labhasetwar Ph.D.b

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Affiliations: a

Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA

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Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic,

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Cleveland, Ohio, USA

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Correspondence to:

Present addresses: 1

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Devon Snow-Lisy Glickman Urological and Kidney Institute Cleveland Clinic Desk Q10-1, 9500 Euclid Ave, Cleveland, OH 44195 Phone 216-445-4473 Fax 216-445-7031 Email: [email protected]

Division of Urology, Ann & Robert H. Lurie Children's Hospital of Chicago,

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Northwestern University Feinberg School of Medicine, Chicago, USA USC Institute of Urology, Catherine and Joseph Aresty Department of Urology, Keck

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School of Medicine of University of Southern California, Los Angeles, CA, USA Conflict of Interest: The authors have nothing to disclose. Running Head: Optimizing nanoparticle delivery to the testicle Keywords: Drug delivery systems, testis, nanoparticles, ligands Word Count: 2,935

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Abstract: Purpose: Nanoparticles, submicroscopic particles typically ranging from 100-300 nm,

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are interesting for the potential treatment of testicular disorders because they can be engineered to allow delivery to privileged tissues such as across the blood-brain-barrier or, theoretically, the blood-testis-barrier. We compare the effects of anatomical and/or ligand targeting on testicular nanoparticle uptake in a rat model.

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Materials and Methods: Rats (n=48) were divided into six groups: control group, intraarterial injection of unconjugated nanoparticles with and without saline flush, intravenous

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injection of unconjugated nanoparticles, intra-arterial injection of follicle stimulating hormone conjugated nanoparticles, intravenous injection of follicle stimulating hormone conjugated nanoparticles and intra-arterial injection of trans-activating transcriptor conjugated nanoparticles. A dose response curve was then assessed for intra-arterially

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injected unconjugated nanoparticles. Using high performance liquid chromatography and histological analysis, nanoparticle uptake by the testicle at 4 hours was determined. Results: Intra-arterial injection resulted in a 5.8 fold increase in uptake as compared to

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intravenous injection at 35 mg/kg of unconjugated nanoparticles (3.7 vs. 0.6 µg nanoparticles/gram testicle p=0.04). Anatomic targeting failed to improve testicular

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uptake in follicle-stimulating hormone conjugated nanoparticles (0.33 vs. 0.38 µg FSHnanoparticles/g testicular tissue for intra-arterial versus intravenous injection respectively p= 0.73). On fluorescence microscopy, nanoparticles were within the testicular interstitium and seminiferous tubules and absent from the testicular vasculature.

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Conclusions: Arterial injection for anatomic targeting nanoparticles to the testis is feasible, improves unconjugated nanoparticle delivery to testicular tissue, and allows

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nanoparticles to cross the gonadal vascular endothelium and blood-testis-barrier.

Introduction:

Nanoparticles (NPs) are submicroscopic particles typically ranging from 100-300

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nm in diameter which can encapsulate novel therapeutics in biodegradable polymer for sustained drug delivery. By encapsulating a drug, these NPs can allow for specific

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tailoring of the drug’s solubility, bio-distribution, and duration of action. NPs are especially interesting for the potential treatment of testicular disorders because their encapsulating polymers and surface characteristics can be modified, therein allowing delivery to specific tissues even across relatively impermeable obstacles similar to the

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blood-testis-barrier such as the brain-blood-barrier.1, 2 Targeted drug delivery to the testes could be used for the treatment of numerous conditions; including but not limited to, the treatment of testicular torsion and infertility associated with elevated reactive oxygen

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species.2-6 At this point, NPs ability to penetrate the testicle has not been evaluated.

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Targeting NPs to an organ or tissue of interest, thereby limiting systemic side effects, can be accomplished by a variety of means including anatomic or ligand targeting. Anatomic targeting implies using features of the anatomy of an organ or tissue of interest to maximize drug delivery. One example of this targeting is to inject the drug into the arterial blood supply of the target organ. A ligand is most frequently a small molecule, select portion of a hormone, antibody, antibody fragment, or peptide that is bound/conjugated to the surface of a NP therein allowing binding of the NP to a site on a

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target. Trans-activating transcriptor peptide (TAT) is one of the most commonly used nonspecific ligands for drug delivery as it non-selectively increases cellular and tissue uptake of conjugated NPs. TAT is a peptide isolated from the HIV virus and is used to

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increase intracellular, intranuclear and tissue delivery across a variety of barriers,

including the blood-brain barrier.7 Another potential ligand to target testicular tissue is a portion of the follicle-stimulating hormone (FSH), a hormone which is uniquely specific

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to the testis. Receptors for FSH are found specifically on the testicular vasculature as well as on Sertoli cells and spermatogonia.8 We have shown NP conjugation to a rationally

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selected portion of FSH increased uptake by and protection of Sertoli cells from oxidative stress.9 The objective of this study is to evaluate the efficacy of anatomic targeting and ligand targeting for the delivery of NPs to testicular tissue in vivo.

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Materials and Methods:

Study Design: This was an in vivo controlled experimental study using rats for which IRB approval was not needed. The Cleveland Clinic Institutional Animal Care and Use

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Committee approved the animal protocol. To compare different methods for targeting NPs to the testis the following experimental groups were evaluated using 35 mg/kg of

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NPs: intra-arterial injected NPs without saline flush and with saline flush, intravenous injected NPs, intra-arterial injected FSH-NPs, intravenous injected FSH-NPs, intraarterial injected TAT-NPs, intra-arterial saline, and intra-arterial control NPs. Sample size was determined with the expectation that intra-arterial injection would have increased variability in uptake requiring 6 animals per intra-arterial injection group, 4 animals per intravenous injection group, and 3 animals per control group. Second a dose

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response curve for intra-arterial NPs was evaluated with 70, 17.5, 8.75, and 4.375 mg/kg NPs (n=4 each group).

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Materials: Poly (D,L-lactide co-glycolide) (PLGA, copolymer ratio 50:50, inherent viscosity 0.87 dl/g) Durect Corporation, (Pelham, AL), Millex-GP 0.22 µm filters

Millipore (Billerica, MA), and Sprague-Dawley rats (SAS-SD) Charles River Laboratory

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(Spencerville, OH) were purchased. Nagase Chemicals (Tokyo, Japan) generously gifted Denacol® EX-521 (MW 742 Da, Pentaepoxy). FSH peptide sequence (Arg-Gln-Cys-His-

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Cys-Gly-Lys-Cys-Asp-Ser-Asp-Ser-Thr-Asp-Cys-Thr-Val-Arg-Gly-Leu) (molecular weight 2184.47) and TAT peptide sequence (Gly-Arg-Lys2-Arg2-Gln-Arg3) (molecular weight 1396.67) were custom synthesized by the Cleveland Clinic Lerner Molecular Biotechnology core (Cleveland, OH). All other chemicals were purchased from Sigma-

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Aldrich (St. Louis, MO).

Formulation of NPs: As previously described, NPs were formulated with a multiple

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emulsion-solvent evaporation technique.9 In summary, 81 mg of PLGA and 9 mg of dimethyl tartaric acid which used as a plasticizer with or without 50 µg of 6-coumarin

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were dissolved in 1.5 ml of chloroform. 6-Coumarin dye was used to analyze location of NPs in testicular tissue qualitatively via fluoroscopic microscopy and quantitatively with HPLC. In 500 µl of purified water, 30 mg of rat serum albumin used as a model protein was dissolved. These solutions were emulsified forming the primary water-in-oil (w/o) emulsion by 2 minutes vortexing and sonication for 2 min on ice at 55W of energy output (XL 2015 Sonicator® ultrasonic processor, Misonix Inc., Farmingdale, NY). A solution

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of 18 ml of 5% w/v poly vinyl alcohol (average molecular weight 30,000-70,000, 87-90% hydrolyzed) solution was prepared by dissolving at 80 °C, cooling to room temperature, filtering through 0.22 µm filter, adding 3-4 drops chloroform and vortexing for 2 min.

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This solution was emulsified into the primary emulsion as above forming the secondary water-in-oil-in-water (w/o/w) emulsion. The secondary w/o/w emulsion was stirred overnight allowing evaporation of chloroform. NPs were recovered and washed by

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ultracentrifugation x 3, 30,000 rpm for 20 min at 4 °C (Beckman OptimaTM: -80K,

Beckman Instruments, Inc., Palo Alto, CA) with resuspension in purified water and

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sonication for 2 min on ice. To eliminate large particles, the solution was centrifuged (Sorvall legend RT, ThermoFischer Scientific, Waltham, MA) at 1,300 rpm for 14 min at 4 °C. Supernatants were frozen at -70 °C for 1 hour and lyophilized (Freeze Dryer, VirTis Company, Inc., Gardiner, NY). NP characterization was performed using NicompTM (380

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ZLS zeta potential particle sizer, Santa Barbara, CA) after dispersing 5 mg of NPs in 2 ml of purified water and sonicating on ice for 30 sec.

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Conjugation of peptide to NPs: NPs were conjugated as previously described by a twostep epoxy method with the first step consisting of activating the NPs with Denacol® and

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the second step where the NPs are conjugated to the ligand/peptide.9, 10 First NPs were suspended by sonication for 30 sec on ice in a 4 ml solution of 50 mM borate buffer (pH 3.0), 12.8 mg of zinc tetrahydrofluroborate hydrate, and 40 mg of Denacol ®. The solution was stirred for 1 hour at 37 °C. NPs were ultracentrifuged x 2 at 30,000 rpm for 20 min at 4 °C and re-suspended in 4 ml borate buffer by sonication on ice for 1 min. For the second step, 10 mg of zinc tetrahydrofluroborate hydrate and 1 mg of peptide were

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added to the NP solution and stirred for 4 hour at 37 °C. NPs were washed of un-reacted peptide using overnight dialysis in 1 L of purified water. NPs were collected by

froze, lyophilized, and characterized as above.

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collecting the supernatant after centrifugation at 1,300 rpm for 10 min at 4 °C. NPs were

Injection of NPs: Male Sprague-Dawley rats (250-400 g) were acclimatized for ≥ 72

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hours prior to experimental use. Following induction with inhaled isoflurane anesthesia (4-5%) the animal was weighed and placed on a heating pad. Isoflurane anesthesia (1-

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3%) was administered via nose cone. After clipping and prepping with betadine and ethanol, eye ointment was applied. For intra-arterial injections, buprenorphine (0.1 mg/kg every 4 to 6 hours) was administered subcutaneously and the skin incision was infused with 0.25% bupivacaine. The abdominal aorta was exposed after midline laparotomy and

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dissected free of the surrounding attachments identifying the bilateral testicular arteries. The aorta immediately superior and inferior to the origin of the testicular arteries was clamped with microvascular bulldog clamps and a 30 gauge bent hypodermic needle

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connected to PE10 tubing (Braintree Scientific, Inc. Braintree, MA) was used to cannulate the clamped aorta. Before and after injection of the experimental NPs or saline,

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0.5 ml saline flush was injected. In the non-saline flush group, animals only had injection of the experimental NP solution. All fluids were administered via infusion pump at 1 ml/min (11 Plus Infusion Pump, Harvard Apparatus, Holliston, MA). First the needle was removed followed by the proximal and then distal microvascular bulldog clamp. Hemostasis was achieved via direct compression of the abdominal aorta puncture site for < 1 minute. The area was inspected confirming hemostasis and incisions were closed,

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fascia with running 4-0 Vicryl and skin with interrupted 5-0 nylon suture (Ethicon Inc. Cincinnati, OH). For intravenous injections the rats were similarly anesthetized. Tail veins were cannulated using a 30 gauge needle and experimental solutions were infused

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(1 ml/min) followed by a 0.5 ml saline flush. After 4 hours the animals were euthanized with isoflurane overdose followed by cervical dislocation to confirm animal death.

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Analysis of testicular uptake of NPs: Quantitative uptake of NPs was evaluated with

HPLC as described previously.11 Organs (testes, brain, kidney, liver and spleen) were

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weighed, homogenized using Tissue-Tearor (Model 985370; Biospec Products, Inc., Bartlesville, OK), frozen, and then lyophilized. On an orbital shaker 6-coumarin was extracted using 3 ml of nitrogen purged methanol (100 rpm for 72 hours at 37 °C). Samples were centrifuged at 4,000 rpm for 10 min at 4°C; 1 ml of supernatant was

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removed and centrifuged at 14,000 rpm for 20 min at 4 °C. That supernatant (900 µL) was then analyzed in triplicate (20 µL) with HPLC (Shimadzu Scientific Instrument, inc. Columbia, MD) using a fluorescence detector Ex(λ) 450 nm/ Em(λ) 490 nm, a mobile

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phase solution of acetonitrile:purified water: 1-heptane sulfonic acid sodium salt monohydrate (65:35:0.005M) and a Nova-Pak® C8 column (2 x 150 mm2 Waters,

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Milford, MA) with 4 µm packing. Uptake of NPs was calculated in µg of NPs per gram of testicular tissue by determining the quantity of 6-coumarin dye in the sample as compared to standard plots multiplied by the weight of tissue homogenized. Standard plots were created with NPs of known concentrations from each formulation. These were processed in a similar fashion using untreated tissue, thereby accounting for minute variations of dye concentration between different NP formulations.

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Qualitative uptake was evaluated with fluoroscopic histologic images. After rinsing tissues in normal saline they were embedded in O.C.T compound and submerged in isopentane over liquid nitrogen (Tissue-Tek, Fisher Scientific, USA). Samples were

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sectioned (10 µm), air dried for 5 minutes, counter-stained with a 10 µg/ml bis-

benzimide, and fixed with 4% paraformaldehyde. Fluoroscopic images were obtained

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using a Leica DMR upright microscope (Leica Microsystems Inc., Buffalo Grove, IL).

Statistical Analysis: Two tailed t tests were used to compare two means, with ANOVA

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being used to compare > 2 means with normal distribution, and Kruskal-Wallis being used to compare > 2 means without normal distribution. Data are expressed as the mean ± the standard error of the mean. Data were excluded if the animal died prior to end point (2) or the sample was lost or contaminated (1/1). P values under 0.05 were considered

Results:

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statistically significant (JMP 9 software, Cary, NC).

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Formulation and characterization of NPs: The mean diameter of NPs dispersed in water (mean hydrodynamic diameter) ranged from 279.4-315.6 nm (Figure 1). A measurement

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of size distribution, the polydispersity index, ranged from 0.047 to 0.099. The NPs all demonstrated a negative charge (zeta potential range -1.02 to -14.10 mV), with intraarterially injected unconjugated NPs demonstrating the most negative charge (zeta potential -14.10 mV) and conjugated NPs demonstrating less negative charges (zeta potential range -1.02 to -5.32 mV).

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Injection technique: Photograph of the animal dissection demonstrates the exposed rat aorta and gonadal arteries (Figure 2 A, B). Due to variations in origin, mobilization of the renal vasculature was needed in some cases to identify the gonadal arteries. NP uptake

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was less without saline flushes for intra-arterially injected unconjugated NPs (0.33±0.16 vs. 3.66±0.99, mean difference 3.34: 95% confidence interval -0.18-6.86 µg NP/g

testicular tissue p=0.06). Left versus right testicular uptake was not significantly different

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(p=0.3-0.9) except in the non-saline flush group where left testicular uptake was 92±7% of the total uptake (p=0.01). No animal was noted to have neurological deficits after cross

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clamp of the aorta. Hemostasis was able to be obtained with pressure in all cases.

NP distribution and testicular uptake: Qualitative evaluation by fluorescence microscopy demonstrated increased NP density in the testicles of anatomically targeted unconjugated

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NPs (Figure 2-C). NPs were noted within the testicular interstitium and outside of blood vessels which were identified by auto-fluorescing subendothelial vascular connective tissue. NPs crossed the blood-testis-barrier with NPs being visualized within

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seminiferous tubules.

Anatomic targeting with intra-arterial injection of unconjugated NPs

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demonstrated significantly increased uptake as compared to all other methods with 3.66±0.99 vs. 0.62±0.19 (intravenous NP, mean difference 3.04: 95% confidence interval 0.23-5.85), 0.33±0.08 (intra-arterial FSH-NP, mean difference 3.33: 95% confidence interval 1.34-5.32), 0.38±0.13 (intravenous FSH-NP, mean difference 3.28: 95% confidence interval 0.70-5.85), and 0.04±0.01 (intra-arterial TAT-NP, mean difference 3.62: 95% confidence interval 0.82-6.41) µg NP/g testicular tissue p=0.01 (Figure 3-A).

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Anatomic targeting failed to change testicular uptake in FSH conjugated NPs (0.33±0.07 vs. 0.38±0.13 µg FSH-NP/g testicular tissue for intra-arterial versus intravenous injection respectively p= 0.73). TAT conjugated NPs showed the lowest uptake of all formulations

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(0.04±0.01 µg NP/g testicular tissue). The dose response curve showed no increased uptake of NPs by the testicle with doses above 17.5 mg/kg, range 3.4-3.6 µg NP/g

testicular tissue (Figure 3-B). The biodistribution of NPs within assayed tissues (testis,

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brain, kidney, liver and spleen) demonstrated that the majority of NPs were taken up by the liver and spleen (range: liver 28-61%, spleen 20-67%, liver and spleen 55-99%).

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Intravenous FSH conjugated NPs demonstrated the highest percentage of testicular uptake (19%) and lowest liver and spleen uptake (55%) as compared to other NP formulations (range: testis 0-1%, liver and spleen 81-99%).

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Discussion:

Here we show that anatomic targeting improves delivery of unconjugated NPs to the testicle. Consistent with previous literature documenting that PLGA is biodegradable

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and biocompatible leading to its extensive use in drug delivery applications, we found no evidence of toxicity.12 Developing strategies to deliver NPs specifically to the testicle

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could have clinical implications for a variety of testicular disorders. One disorder where anatomic targeting would be most useful is testicular torsion, where the gonadal vasculature is easily accessible by the surgeon and current management consisting of surgical de-torsion only allows salvage of the affected testicle in 64% of cases.13 Most work with NPs has focused on cancer treatment as the impacts of systemically administered chemotherapies are especially toxic and tumor targeting is

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simplified due to increased tumor vascular permeability.14 Here we evaluate other targeting techniques which could be useful in benign pathologic processes. We hypothesized that anatomic targeting would improve NP uptake and that FSH would be a

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valuable targeting ligand. FSH receptors are present in the rat testicular vascular

endothelium where they rapidly (within 15 minutes) bring FSH into the testicular

interstitium.15 A segment of rationally selected FSH hormone, which has previously been

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shown to stimulate estradiol synthesis of rat Sertoli cells, when used as a ligand

facilitated NP uptake by mouse Sertoli cells in vitro.9, 16, 17 Interestingly, in vivo we

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found that anatomically targeted unconjugated NPs had the highest uptake. This could be due to small differences in NP shape, size, and surface charges that occur with conjugation.18, 19 These changes may have been significant enough to change the biodistribution of the NPs and/or limit the efficacy of ligand targeting.

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We found that intravenously injected FSH conjugated NPs had similar uptake to intravenously injected unconjugated NPs and intra-arterially injected FSH conjugated NPs. This suggests that conjugation did improve intravenous delivery of FSH conjugated

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NPs. These findings were not replicated with intra-arterially injected TAT conjugated

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NPs which had the lowest uptake of all formulations tested. The biodistribution, as identified by comparing uptake in the testicle to the liver, spleen, kidney, and brain showed intravenously injected FSH conjugated NPs had a higher percentage of testicular uptake and lowest liver and spleen uptake as compared to other NP formulations (19% testis uptake, 55% liver and spleen uptake as compared to 0-1% testis uptake and 81-99% liver and spleen uptake). Evaluation of the complete biodistribution of FSH conjugated NPs at different time points would further elucidate the mechanism of these findings.

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While qualitatively no NPs were noted within the testicular microvasculature, a limitation is that we were unable to quantify the amount of NPs within the testicular vasculature, interstitium, versus the quantity that crossed the blood-testis-barrier.

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Qualitatively NPs cross the blood-testis-barrier with both anatomic targeting and FSH ligand targeting, as demonstrated by visualization of NPs within the lumens of

seminiferous tubules. Though there is a large degree of homology, there are few

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differences between the rat and human blood-testis-barrier. Reported differences include a maintained blood-testis-barrier with cryptorchidism in rats as compared to a loss of the

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barrier in humans, fenestration of human testicular endothelial cells as compared to unfenestrated rat endothelial cells, and differential expression of occludin in the tight junctions between Sertoli cells in rats and humans.20-25 While most of these differences lead one to believe penetration of NPs past the blood-testis-barrier would be greater in a

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human patient, if the mechanism relies upon occludin, the opposite may be true. The mechanism by which NPs cross the blood-testis-barrier has not been elucidated in this study and could be of further interest. By allowing for NP delivery to the lumen of the rat

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seminiferous tubule, this study provides a stepping stone for antioxidant loaded nanoparticle treatment of infertility associated with elevated reactive oxygen species

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including idiopathic infertility and varicocele.26

Conclusions:

NP delivery to the testicles is feasible and allows for delivery across the bloodtestis barrier with anatomic targeting of unconjugated NPs demonstrating a 5.8 fold increase in testicular tissue delivery. This study represents an important step towards

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utilization of NP treatments for a variety of testicular disorders including, most notably, testicular torsion where the testicular vasculature is easily accessible by the surgeon at

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time of detorsion.

Acknowledgements:

The authors would like to thank the Research Program Committee of the

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Cleveland Clinic for financial support. The authors have no financial disclosures to

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report.

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References: 1.

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Figure Legends: Figure 1: NPs show uniform size distribution and negative charge NP characteristics for different formulations. NPs showed consistently negative charges

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(range -1.02 to -14.10 mV) with uniform sizes (range 279.4 to 315.6 nm). Example

particle size distribution as measured using Nicomp analysis (Intra-arterial TAT-NP).

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Figure 2: Intra-arterial injection technique demonstrates NP uptake into the testicular parenchyma and across the blood-testis-barrier.

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Photograph A demonstrates surgical dissection with the left renal vasculature (RV-renal vein) looped by 3-0 silk suture. Rat gonadal arteries (*) demonstrated variable origins. Photograph B demonstrates sample injection with microvascular bulldog clamps across the aorta proximally and distally with a bent 30 gauge needle being used to access the

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aorta at the level of the gonadal artery (*).

Photomicrogaph C demonstrates NP uptake by testicular parenchyma outside of blood vessels (BV) and within the lumen of the seminiferous tubules (#) of intra-arterially

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injected NPs. 6-Coumarin in NPs fluoresces green. Cellular DNA, stained with bisbenzimide, fluoresces blue. DNA condenses in the head of spermatids (#) indicating

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the lumen of the seminiferous tubule.

Figure 3: Intra-arterial injection increases testicular uptake of unconjugated NPs. Intra-arterial injection of unconjugated NPs optimized testicular uptake with saturation of uptake demonstrated above 17.5 mg/kg NPs injected.

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A: Uptake is significantly increased by 5.8 fold with anatomic targeting of unconjugated NPs (3.66±0.99 vs. 0.62±0.19 µg NP/g testicular tissue for intra-arterial versus intravenous injection respectively p= 0.04 (*), mean difference 3.0 µg NP/g testicular

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tissue: 95% confidence interval 0.23-5.85). FSH conjugated NPs demonstrate equivalent testicular uptake regardless of anatomic targeting (0.33±0.07 vs. 0.38±0.13 µg FSH-NP/g testicular tissue for intra-arterial versus intravenous injection respectively p= 0.73). TAT

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conjugated NPs demonstrate low (0.04±0.01 µg NP/g testicular tissue) with anatomic targeting. Background signal for controls (saline and NPs without dye) were 0.

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B: Dose response curve for intra-arterially injected unconjugated NPs demonstrated

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optimized level of NP uptake at doses > 17.5 mg/kg.

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Abbreviations: Nanoparticles (NPs), Trans-activating transcriptor peptide (TAT), folliclestimulating hormone (FSH) high performance liquid chromatography (HPLC), Poly (D,L-lactide

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co-glycolide) (PLGA)