# Testes microstructure and hormonal changes after administration of silver nanoparticles loaded with disoproxil tenofovir in mice with type 2 diabetes.

### moral statement

Prior to initiating this research, ethical approval was requested and received from the Animal Ethics and Research Committee (AREC) of the University of KwaZulu-Natal (UKZN) with approval number AREC/043/019D. This study was conducted at the Biomedical Resource Unit (BRU), UKZN, Westville Campus, under the Laboratory Animal Care Principles of the UKZN approved guidelines and the 3Rs principle. Also, this study follows the recommendations in the Guidelines for Research on Animals: Reporting of Experiments in Vivo (ARRIVE), and the animals received humane care.

### Chemicals and drugs

Tenofovir disoproxil fumarate (TDF), a drug of the nucleoside reverse transcriptase inhibitor (NRTI) class of antiretroviral drugs at a human equivalent dose of 300 mg, was purchased from Dis-Chem Pharmacy, Ballito, South Africa. Fructose (Frt), streptozotocin (STZ), trisodium citrate (TSC), sodium hydroxide (NaOH), and silver nitrate (AgNO)3) of Analytical Grade from Sigma-Aldrich Corporation, Johannesburg, South Africa.

### AgNP . synthesis

This study used AgNO3 As a precursor and TSC to reduce and stabilize the synthesized AgNPs30. Exactly, 0.03 mol/dm3 Mixed well with 0.1 mol/dm aqueous3 TSC, and the resulting solution was under constant stirring for 5 min at 90 °C, and NaOH (pH 10.5) was used to change the pH for 90 min. A color change from colorless to amber yellow is observed. The synthesized AgNP was cooled at room temperature, centrifuged at 12,000 rpm for 15 min, and oven dried at 40 °C overnight.31. Then, 0.35 M TDF was mixed with 100 mL of synthesized AgNP, and the mixture was under continuous stirring in ultra sonication to ensure proper interaction of the components (TDF and AgNPs).

TDF-AgNP was centrifuged at 4500 rpm, for 40 min at 40 °C to separate the unincorporated drug. The obtained supernatant was analyzed with a UV spectrophotometer at a wavelength of 274 nm to calculate the amount of unincorporated drug (W1) from the total amount of drug coupled with silver nanoparticles (W2). The percentage drug incorporation efficiency (% IE) was estimated using the equation below32.

$$text{Percent Efficiency Combined} (%text{IE}): =frac {text{TW} 2-text{TW}1}{text{TW}2}times 100 %$$

(1)

TW2 is the total amount of TDF loaded with silver nanoparticles, and TW1 represents the amount of unincorporated TDF. Compound AgNP and TDF-AgNP coupling were confirmed using ultraviolet visible (UV-Vis) spectroscopy (Shimadzu MultSpec-1501, Shimadzu Corporation, Tokyo, Japan). Also, the FTIR (Perkin-Elmer Universal ATR Spectrometer, USA) confirmed the functional groups and the bonds. The morphology and size of the synthesized nanoparticles were investigated by a high-resolution electron microscope (HR-TEM, JEOL 2100, Japan) operating at 200 kV and field-emission scanning electron microscopy (FESEM, Carl Zeiss, Germany) performed at 5 kV. Also, the elemental components were confirmed by energy-dispersive X-ray (EDX, Aztec Analysis Program, England).

### experimental design

A total of thirty-six (n = 36) adult male Sprague-Dawley rats weighing between 230-250 g were used for this research. Mice were housed in ventilated plastic cages (3 mice per cage with dimensions 52 cm x 36 cm and 24 cm, length, width and height, respectively, with soft sawdust bedding). Mice were maintained in standard animal house conditions at temperatures from 23 to 25 °C; 12 hours of natural light daily. Mice were fed standard rat pellets from Meadow Feed, Division of Astral Operations Limited, Durban, South Africa, and water was administered as ad libitum.

After 2 weeks of acclimatization, the initial body weights of the mice were recorded, and then the mice were randomly divided into diabetic groups (n = 18) and a non-diabetic group n = 18). Rats assigned to the diabetic group received fructose (Frt) and streptozotocin (STZ) to induce T2DM, according to a protocol previously described by Wilson and Islam.33. Based on this model, 10% frt was dissolved in ad-libitum drinking water for 2 weeks to cause insulin resistance. Then, mice were induced with 40 mg/kg freshly prepared STZ in 0.1 M citrate buffer (pH 4.5) based on body weight. More so, mice in the non-diabetic group received the same volume of 0.1 M citrate solution (pH 4.5) intraperitoneally. The diabetes model was characterized by checking for hyperglycemia by fasting blood glucose using glucometers and chips (Acucheck).(s)Boehringer Mannheim, Germany). Experimental rats with a blood glucose level of 200 mg/dL and above were considered to have diabetes and were included in the diabetic group. The diabetic and non-diabetic groups were divided into three groups for each group (n = 6) as follows.

Group 1 represents non-diabetic (NDC) mice – receiving 1 mL of distilled water (po) daily.

Group II refers to non-diabetic tenofovir (NDT)-treated rats treated with 26.8 mg/kg/body weight per day of tenofovir (r).

The third group represents non-diabetic TDF-AgNP (NDNT) mice given 6.7 mg/kg/body weight TDF-AgNP (daily i.p.).

Group IV represents diabetes control (DC) rats – receiving 1 mL of distilled water (po) per day.

Group V reported diabetic rats (DT)-treated with 26.8 mg/kg/body weight daily tenofovir (R).

The sixth group represents diabetic TDF-AgNP (DNT) mice given 6.7 mg/ml/body weight TDF-AgNP daily (IP).

### animal treatment

The tablet form of TDF (300 mg) was used in this experiment. The tablets were crushed into a powder form, and adequate amounts were weighed following the recommended human doses. The animal dose was calculated from the human equivalent dose (HED) using the US Food and Drug Administration (FDA) recommended formula.34, 35;

$${text{HED}}left(frac{{text{mg}}}{{text{kg}}} right) = frac {{text{Animal NOAEL}}left( frac {{text {mg}}} {{text {kg}}} right) times {text {Animal Weight}} left ({text {kg}} right)} {{ text {human weight}}left ({text{kg}}right) ^{1-0.67}}.$$

Rats in the treatment groups were given 26.8 mg/kg/day of TDF based on this formula.

The experiment lasted for thirteen weeks, while the administration lasted for 56 days. More so, comprehensive environmental enrichment practices and the application of 3Rs and five liberties for pain, discomfort, and distress during the experiment were considered. After 3R was applied, daily administration in groups 3 and 6 through the peritoneum was reduced to 6 days per week. Animals were killed by excessive inhalation of isoflurane 24 hours after the last administration on day 57. Blood samples were collected by intracardiac puncture in different sample bottles, and testes were excised for different analyses.

### Determining the number and viability of sperm

Sperm count and viability were analyzed as previously described36. Shortly after euthanasia, the caudal epididymis was removed from the experimental mice and collected in 5 ml of normal saline in each labeled bottle. 2 ml was withdrawn from it and used to mince 1 tail of the epididymis into a Petri dish. This dilution factor (2 mL) remained constant throughout and for all mice. Then, the epididymal fluid was mixed well, and approximately 20 μl of this diluted sample was transferred to each of the counting chambers of the hemocytometer and counted using Bio-Rads Equipment (TC20 machine). Both sides of the counting chamber were used for each sample and the average was recorded to the nearest million/mL37. Sperm viability was assessed by staining a dry smear of sperm on a glass slide with eosin-nigrosine staining after observation under a light microscope (Leica DM 500) at 400× magnification.

### reproductive hormones analysis

Blood samples were collected for the hormonal assay through a cardiac puncture after the animals were euthanized. Blood samples were transferred to serum bottles, allowed to stand for 30 min and centrifuged at 3000 rpm for 15 min in a Beckmann bench centrifuge. According to the manufacturer’s instructions, the serum concentration of reproductive hormones was determined using enzyme-linked immunosorbent assay (ELISA) kits (Elabscience). Serum levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were determined using rat ELISA kits (Elabscience) with catalog numbers: E-EL-R0597, E-EL-R0391 and E-EL-R0026 and E-EL-R0389 for GnRH, FSH, LH and testosterone, respectively, by manufacturer’s protocols. Sample analyzes were performed in duplicate, and values ​​were estimated from the resulting standard curve from the calculated concentrations.

### Determination of the expression of insulin-like growth factor 1 (IGF-1)

An equal volume of 0.5 g of each harvested testes was homogenized using a 5 ml sodium phosphate buffer buffer with 1% Triton X-100 (50 mM, pH 7.5). Testis homogenates were centrifuged at 20,000 g in a Centrikon H-401 centrifuge (Germany) for 10 min at 4 °C. Then, the supernatants were collected, poured into 2 ml Eppendorf tubes, labeled and kept at −80 °C until further analysis. Testicular homogenates for IGF-1 level were determined using a dual-antibody ELISA kit (ELISA) with catalog number E-EL-R0010, Lot: E3YXQMELZQ following the manufacturer’s protocol.

### Electron microscopy procedure

The testicle was cut into 1 mm3 Immediately fixed sections in modified Karnovsky’s fixative containing 0.2 M cacodylate, 0.2% picric acid, 4% paraformaldehyde, 0.02% calcium chloride, and 4% glutaraldehyde. Then, the tissues were dried in graded acetone solutions and incorporated into resin (Araldite). A KOS microwave tissue processor was used for post-embedding procedures such as rinsing, post-fixation, dehydration, and infiltration. Also, 1 μm thick semi-thin pieces were cut with a glass knife using a Leica Ultracut R Ultramicrotome. Tissues were stained with 1% toluidine blue dissolved in 1% borax for 30 min at 40–50 °C. Then, the stained tissues were washed, dried, and examined with a general orientation light microscope. Leica Ultracut R Ultramicrotome (Leica Microsystems, Milton Keynes, England) was used to obtain tissue sections less than 100 nm thick. Grids were then stained with 2% alcohol uranyl acetate for 10 min in the dark, washed thoroughly in sterile water, and air dried before assay. The sections were then viewed and imaged using a FEI TECNAI™ ​​(TEM) transmission electron microscope (120 kV).38. Furthermore, TEM was performed at the Microscopy and Microanalysis Unit (MMU), University of KwaZulu-Natal, Westville, South Africa.

### statistical analysis

In this study, the statistical tool used was one-way analysis of variance (ANOVA) to determine the mean differences between groups. Also, Tukey’s multiple comparison post hoc tests were performed using Graph pad prism®, statistical software version 7.0. The results were visually displayed and expressed as mean ± standard error of the mean (SEM) with a confidence level of 95% (s< 0.05).

### Ethical Consent and Consent to Share

Ethical permission for this study was obtained from the Animal Ethics and Research Committee (AREC) of the University of KwaZulu-Natal with approval number AREC/043/019D. Also, this study was conducted at the Biomedical Resources Unit (BRU) of the University of KwaZulu-Natal, Westville Campus, under the Laboratory Animal Care Principle of the University of KwaZulu-Natal Standard Approved Guidelines.