Identification and validation of Sertoli cell homing peptides as molecular steering for testis targeted drug delivery

Abstract The testicle, an organ privileged with immunity because of Blood-Testis Barrier (BTB), poses a major impediment to developing and delivering drugs to the testes. These problems can be prevented by targeting testicular cells using specific ligands, such as homing peptides. This is the first study to demonstrate the successful selection of Sertoli cell homing peptides using a phage display peptide library. The identification of peptides is performed with Sanger sequencing and high-throughput NGS. The Sertoli cell and testis targeting potential of the SCHP1 and SCHP2 was confirmed using confocal microscopy and flow cytometry of the FITC-labelled peptides and in vivo bio-distribution of the corresponding Cy5.5-tagged peptides. Secondary structures were predicted in the setting of different polarity by circular dichroism. The results suggest that SCHP1 and SCHP2 can effectively target Sertoli cells. In vivo bio-distribution in mouse models indicated significantly higher uptake of SCHP1 and SCHP2 by testes compared with the heart, brain, and spleen. SCHP1 and SCHP2 can be adopted as molecular steering for targeted male contraceptive delivery, treatment of testicular cancer, and male infertility. Further development of the peptides into peptidomimetics may increase their stability, and information on the molecular targets of these peptides may reveal their therapeutic potential.


Introduction
Germ cells, Sertoli cells, and Leydig cells are important components of the testis. Sperm production occurs in seminiferous tubules with the help of Sertoli cells, and androgen production occurs between seminiferous tubules by Leydig cells. Various disorders are associated with these testicular cells, including infertility, testicular cancer, and oxidative stress. Male factor infertility accounts for nearly 40% of 8-10% of infertile couples worldwide and 3.9-16.8% in India. 1 Male infertility could be because of various reasons, including endocrine, iatrogenic, congenital abnormalities, acquired testicular damage, varicocele, oxidative stress, male accessory gland infection, genetic or idiopathic [1]. Current management strategies for male factor infertility include drug treatment, depending on the cause of infertility and artificial reproductive technologies. Drug treatment includes hormonal treatment, antioxidant treatment, and a combination of both [2,3]. Systemic delivery of exogenous testosterone to treat male infertility result in feedback inhibition, ultimately worsening the condition [4]. Testicular cancer accounts for 1% of all men globally, but it is the most common malignancy between the age group of 15 to 35 [5]. Germ cell cancer accounts for approximately 90 to 95% of testicular cancers [6]. Current treatment strategies include orchiectomy, active surveillance radiotherapy, chemotherapy, and RPLND-Retroperitoneal Lymph node dissection [7,8]. Chemotherapy plays a significant role in the treatment of germ cell tumours with an approximate 95% cure rate, but there are various complications, such as systemic toxicities, cardiovascular effects, and secondary malignancies. These treatment-related toxicities could be acute or chronic with long-term effects [9]. The people are now accepting the active role of a male partner in family planning, but male contraceptives are limited to condoms and vasectomy. Vasectomy is non-reversible, and condoms have marginal efficacy [10]. Testosterone has been used as a reversible male contraceptive that acts via a negative feedback mechanism, but being a hormone, it affects the normal sexual functions of the individual. Researchers are now focussing on non-hormonal instead of hormonal contraceptives because it will not affect testosterone concentration and normal sexual functions of the individual. A few examples of non-hormonal contraceptives include adjudin, H2-Gamendazole, eppine-based drugs etc. [11]. In recent years, developments have been made in non-hormonal contraceptive drugs such as adjudin, but the delivery of this drug faces the same problems, such as a meagre margin between safety and efficacy [12].
Conventional treatment and delivery methods of drugs for testicular disorders face limitations such as low efficacy because of the need for high doses and high dosing frequency, systemic toxicities, the problem of feedback inhibition, etc. All these issues can be solved with the help of selective targeting of drugs to the testis. Drug targeting to testis to treat testicular pathologies will be more advantageous than conventional drug delivery methods. Targeted drug delivery assists the drug molecules in accumulating at the desired site or tissue and reduces the systemic side effects. Passive targeting is based on anatomic or physiological factors and active targeting utilises the targeting ligands for the delivery of the drugs [13]. Depending upon the type of receptor expressed or overexpressed, the location and nature of the cell, and the metabolism profile of a particular cell or pathological condition, they have specific tags called zip codes [14]. These zip codes can be exploited to differentiate between target and non-target tissues or cells for identifying targeting ligands. Peptides have shown promising properties as ligands. They have various advantages over other ligands, such as high specificity, low immunogenicity, ease of modification, low cost, rapid clearance from non-target sites. [15]. Phage display peptide libraries provide an excellent platform for selecting such peptide ligands using the procedure called bio-panning. The technique was first described by George P. Smith in 1985, in which a DNA encoding for a specific peptide sequence is incorporated into the bacteriophage genome, leading to the expression of the peptide on the bacteriophage coat [16]. People have identified peptide ligands targeting various cancers and normal organs, including the brain, kidney, skin, pancreas, prostate, breast tissue, human umbilical cords, adipose tissue, and heart [14,[17][18][19][20][21][22][23][24][25]. RGD and NGR peptides recognising integrin and aminopeptidase N are the tumour-targeting peptides. Peptides identified using a phage display libraries are being practiced to deliver drugs across blood tissue barriers, such as the blood-brain barrier and gastrointestinal tract [26]. Homing peptides are highly selective and potent hence provide good efficacy, safety, and tolerability. They are cost-effective and easy to manufacture and modify [27]. Novel peptides targeting Sertoli cells can open new avenues for targeted drug delivery to the testis to deal with various testicular pathologies.
Targeting testicular cells and tissue is important to overcome the hurdle the Blood-Testis Barrier (BTB) provides in developing and delivering drugs to treat testicular anomalies. Almost every testicular pathology or contraceptive drug is cell-specific or targets cell-specific molecules, but there is no testicular cells targeting ligand available to date. Somatic cells like Sertoli and Leydig cells, and also the germ cells can be targeted for treating or delivering drugs for specific testicular pathologies [28]. Sertoli cells form the blood-testis barrier, and the process of spermatogenesis occurs in the compartment protected by this barrier; hence, Sertoli cells provide the potential target for the development and delivery of male contraceptive drugs [29]. Antioxidants such as superoxide dismutase targeting to Sertoli cells can help to address the problem of intratesticular oxidative stress [28,30]. Ligands selectively targeting Sertoli cells may open new avenues for treating various testicular disorders. In this study, we have attempted to identify Sertoli cell homing peptides for testicular targeting using a phage display peptide library. The Sertoli cell homing peptides (SCHPs) may provide better opportunities in the future for the targeted delivery of drugs and male contraceptives to the testis.

Phage display library
Ph.D.-12, a combinatorial phage display library of random 12-mer peptides fused to a minor coat protein (pIII) of the M13 phage, was purchased from New England Biolabs. The displayed peptide is expressed at the N-terminus of pIII. The library consists of approximately 10 9 unique sequences. 10 µL of the library was used to start the experiment, which contained approximately 100 copies of each peptide.

Cell culture
TM4 (Mouse Sertoli cells) (ATCC CRL 1715) were obtained from an American-type culture collection, and Dr. Susan Thomas of Bioinformatics Centre ICMR-NIRRCH gifted HEK293. TM4 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) Nutrient Mixture F-12 supplemented with 5% horse serum and 2.5% foetal bovine serum, and HEK293 cells were cultured in DMEM Nutrient Mixture F-12 (Ham) (1:1) supplemented with 10% foetal bovine serum. Both the cell lines were maintained at 37° C with 5% CO2.

Animal use and care in the study
All the procedures performed on adult Balb/C male mice (6-8 weeks old) were approved by the Institutional Animal Ethics Committee of ICMR-National Institute for Research in Reproductive and Child Health Mumbai (IAEC-03/20).

In-vitro biopanning
Mouse Sertoli cells (TM4) were seeded at the density of 0.5 x 10 6 cells/well in 6 well plate before 48 h of biopanning. The spent medium was removed on the day of biopanning, and DMEM/F12 w/o serum was added to the wells. After 1 h incubation at 37 °C with 5% CO2, biopanning solution containing 1*10 11 phages 10 µL of PhD-12 library, protease inhibitor, and chloroquine in PBS (Phosphate Buffered Saline) with 0.1% BSA (Bovine Serum Albumin) was added and incubated for 1 h. unbound phages were removed by washing four times for 5 min each with 0.1% BSA in PBS. The cell surface homing phages were eluted with two washes of 1 ml of 0.1 M HCl-Glycine, pH 2.2 + 0.9% NaCl for 5 min. Again, the cells were washed with 0.1% BSA in PBS twice, and 1 ml of 30 mM Tris-HCl, pH 8.0, was added and incubated on ice for 30 min, and cells were frozen at −20° C overnight. The next day, cell-penetrating phages were collected from the disrupted cells. Enrichment of the Sertoli cell surface homing and Sertoli cell-penetrating phages was achieved with three rounds of biopanning [31].

In-vivo biopanning
Amplified Sertoli cell surface homing phage pool and Sertoli cell-penetrating phage pool from the 3 rd round of in-vitro biopanning were used to perform in-vivo biopanning. Briefly, 1*10 11 Phages in 100 µL of DMEM medium were injected into Balb/C mice intravenously and allowed to circulate for 30 min. Mice were sacrificed, and all vital organs were collected and stored at −80 °C. The next day, testis was minced in DMEM medium, and phages were recovered and amplified for the next round of biopanning. Enrichment of testis homing phages was achieved through three rounds of such in-vivo biopanning [32].

Phage titration and amplification during biopanning
Phage titration was performed with the qPCR standard curve method as described earlier by Peng et al. [33], tenfold concentrations of M13 ssDNA from 0.1fg/µL to10 6 fg/µL (2.689*10 1 gc/ µL to 2.689*10 8 gc/µL) were used as standards. Samples were precipitated with PEG (Polyethelene glycol)/NaCl for M13 phage isolation and treated with DNase I at 37 °C for 10 min, followed by heat denaturation at 100 °C; these samples were used as a template for qPCR. The reaction mixture of 10 µL consists of 5 µL SYBr green Power up 2x MM, 0.5 µL (10 µM stock) forward primer and reverse primer each, 2 µL nuclease-free water, and 2 µL template ssDNA (standard/sample). Primers used are forward primer 5′-CAC CGT TCA TCT GTC CTC TTT-3′ and reverse primer 5′-CGA CCT GCT CCA TGT TAC TTA G-3′. Reaction conditions were 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, melt curve at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The reaction was performed on Applied Biosystems's QuantStudio5 Real-time PCR and analysed with QuantStudio TM Design and Analysis software.

Plaque assay and Sanger sequencing
Plaque assay was performed according to the New England Biolab's instruction manual. Blue-colored plaques were picked and amplified in E. coli ER2738 host cells. Amplified phage clones were isolated with PEG/NaCl precipitation, and ssDNA was isolated according to the protocol mentioned by Green et al. [34]. PCR was performed to amplify the product using forward primer: 5′-TGG TTG TTG TCA TTG TCG GC-3′ and reverse primer: 5′-GCA AGC CCA ATA GGA ACC CA-3′. PCR reaction mixture of 50 µL consisted of 25 µL 2x DreamTaq Green PCR Master Mix, 2.5 µL each of forward and reverse primer (10 µM stock), 15 µL nuclease-free water, and 5 µL template. PCR conditions were initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and Final extension at 72 °C for 5 min. The reaction was performed on Agilent's Sure Cycler 8800. The PCR product was run on 1.8% agarose gel, extracted using PureLink Quick gel extraction kit; Sanger sequencing was performed with -96gIII reverse sequencing primer provided in the PhD-12 phage library kit.

Next-generation sequencing of phage pool
Phage pool from the 3 rd round (last round of in-vitro panning) and the 6 th round (last round of in-vivo panning) were subjected to NGS. Phage DNA (ssDNA) was isolated from the phage pools as described by Green et al. [34], and PCR was performed to prepare 77 bp amplicon (Supplementary Figure 2) for Illumina sequencing; the primers and barcodes used were picked from Matochko et al. [35], forward primer 5'CCT TTC TAT TCT CAC TCT3' , reverse primer 1 with barcode 5'TTC CGA TAA CCC GAA CCT CCA CC3' and reverse primer 2 with barcode 5'CTG ACC GAA CCC GAA CCT CCA CC3' . The reaction mixture consisted of 25 µL OneTaq® Hot Start Quick-Load® 2X Master Mix, 5 µL of each forward and reverse primer, and 5 µL of template ssDNA. The reaction was performed at 95 °C for 3 min, 35 cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 20 s, elongation at 72 °C for 30 s, and final elongation at 72 °C for 5 min. The PCR product was run on 2% agarose gel and was extracted using GeneJet gel extraction kit. Polyacrylamide gel electrophoresis (10%) was performed with 2 µL of the final product long with low range DNA ladder. The final 77 bp PCR product was further submitted for Illumina sequencing at Bioxplore labs, Chennai, India.

Confocal microscopy of M13 bacteriophage
The homing potential of the SCHP1 and SCHP2 phages to TM4 cells was analysed with an anti-M13 antibody. Briefly, 0.2*10 6 cells were seeded on pre-sterilized coverslips in 12-well plates and allowed to attach for 24 h. These cells were incubated with 1*10 11 SCHP1/SCHP2 phages for 1 h. After incubation, the cells were washed three times in DPBS w/Ca, Mg (Dulbecco's phosphate-buffered saline with calcium and magnesium), and fixed for 10 min with 4% paraformaldehyde at room temperature. Blocking was performed in 1% BSA for 1 hand incubated with an anti M13 antibody (Cat# MA1-06604) at 4 °C overnight. The next day, primary antibody was removed, cells were washed three times, 5 min each with DPBS, and a secondary antibody labelled with Alexa Fluor 594 was added for 1 h. After 1 h incubation, cells were washed three times with DPBS, and the coverslips were mounted on clean slides with vectasheild reagent and observed at NIRRCH core confocal microscopy facility.

Confocal microscopy of peptides
Briefly, 0.1*10 6 cells were seeded on a pre-sterilised coverslip in 12-well plates and allowed to attach overnight. The next day, FITC labelled SCHP1, SCHP2 were added per well and incubated for 1 h. Cells were washed three times with DPBS w/Ca Mg, fixed with 4% PFA (Paraformaldehyde), and counter-stained with DAPI for the nucleus and rhodamine-phalloidin (Invitrogen Cat#R415) for the cytoskeleton. Slides were prepared and analysed with confocal microscopy.

Flow cytometry analysis of peptides
Briefly, 0.5*10 6 cells were seeded per well in 6-well plates and allowed to attach overnight. The next day, cells were treated with different concentrations of the FITC labelled SCHP1 and SCHP2 for 1 h. After the incubation, cells were detached with 1 mM EDTA solution, washed three times with DPBS, and analysed with the flow cytometer.

MTT assay
Cytotoxicity of the SCHP1 and SCHP2 peptides was assessed with MTT assay. Briefly, 0.05*10 6 TM4 cells were seeded per well of 96-well plates and allowed to attach overnight. The next day, the cells were treated with two-fold increasing concentrations of SCHP1/ SCHP2 peptides for 48 h. After 48 h incubation, the spent medium was removed, and MTT solution was added at 50 µg/well and incubated for 4 h at 37 °C. MTT solution was removed, and 100 µL of DMSO (Dimethyl sulfoxide) was added to each well and incubated for 30 min at room temperature. OD was taken at 570 nm, and the percent cell viability was calculated.

Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy was performed on a Jasco J-810 spectropolarimeter using a 1-mm cuvette. The peptides were dissolved in different concentrations, i.e. 10%, 25%, 50%, 75%, and 90% of trifluoroethanol (TFE) in water at a concentration of 0.5 mg/ mL. Spectra were collected every 2 nm from 260 to 190 nm. The CD spectra are reported as Molar ellipticity.

Plasma stability study
SCHP1/SCHP2 unlabelled peptide, FITC labelled and Cy5.5 labelled was dissolved in 10 µL of DMSO followed by 1 ml of25% mouse plasma in DMEM medium. The mixture was incubated at 37oC and 100 µL of the mixture was taken at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6hand 6htime point. Plasma protein of the reaction mixture was precipitated with the addition of 200 µL of 95% chilled ethanol followed by vortex and centrifugation at 13000 g for 2 min at 4oC. The supernatant was collected and 60 µL of it was injected to C18 column to study the stability of the peptide over the period of 8 h. For the mobile phase of RP-HPLC, a mixture of water (0.1% TFA) and acetonitrile (0.1% TFA) in a linear gradient of 25-50%, 25-65% and 25-80% acetonitrile in 20 min was used for unlabelled, FITC labelled and Cy5.5 labelled peptides respectively. uV detection wavelengths were set at 220 nm, 541 nm, and 675 nm for unlabelled peptides, FITC labelled and Cy5.5 labelled peptides, respectively. Stable peptide percentage at different time point was determined by considering 0 min peptide concentration as 100% peptide, and the test was performed in triplicates.

In-vivo imaging
SCHP1 and SCHP2 conjugated to Cy5.5 NIR dye were obtained from LifeTein LLC for in vivo imaging using IVIS lumina III in-vivo imaging system. Adult male Balb/C mice (6-8 weeks old were randomly distributed in three groups (n = 9), i.e. SCHP1, SCHP2, and free Cy5.5 dye. Cy5.5 labelled SCHP1 and SCHP2 were injected into mice intravenously at 20 µg in 100 µL PBS. Mice were dissected, and vital organs, including testis, were collected after 1 h, 6 h, and 24 h. The organ imaging was performed using Perkin Elmer's IVIS lumina III small in-vivo imaging system, and images were analysed with Living Image software.

Screening of Sertoli cell homing peptides
The PhD-12 phage display peptide library was used to screen the Sertoli cell homing peptides. Initially, in two separate sets of experiments, three rounds of bio-panning were performed on TM4 mouse Sertoli cells to get SCSHPs (Sertoli cell surface homing peptides) and SCPPs (Sertoli cell-penetrating peptides). The enriched SCSHP and SCPP phage pool obtained after the 3 rd round of bio-panning with the TM4 cell line was used as input for two separate in-vivo bio-panning in a mouse model, and three rounds of in-vivo bio-panning were performed. After the 6 th round of bio-panning, i.e. the 3 rd round in the mouse model, we got enriched SCSHP and SCPP pools from the testis. M13 bacteriophage titration after each round indicated an enrichment of the phage pool in 3 rd and 6 th rounds of bio-panning for screening of SCSHPs and SCPPs ( Figure 1A).

Identification of Sertoli cell homing peptides
Sertoli cell homing peptide identification was performed using Sanger sequencing and next-generation amplicon sequencing. A plaque assay was performed for the phage pool obtained after the 3 rd and 6 th rounds of bio-panning for screening of SCSHPs and SCPPs. PCR was done using M13 phage ssDNA as a template to amplify the product for Sanger sequencing. PCR products of 416 bp (Supplementary Figure 1) from 277 phage clones were subjected to Sanger sequencing; after removing sequences w/o 36 bp insert, 254 total peptide sequences were obtained, of which 137 were unique peptides (Supplementary Table 1). Depending upon the frequency of the peptides in the SCSHP and SCPP phage pool, these were named SCHP1 to SCHP137, and the top 18 SCHPs were found more than once in either of the pool from 3 rd or 6 th rounds. The overall summary of the peptide obtained with Sanger sequencing is shown in Table 1.
Phage pools from the 3 rd round (last round of in-vitro panning) and the 6 th round (last round of in-vivo panning) from the SCSHP and SCPP biopanning were subjected to high throughput sequencing. The number of unique peptides normalised with a total number of good reads for both the SCSHP and SCPP panning experiments was decreased in the 6 th round compared to the 3 rd round ( Figure 1B), which indicates enrichment of a few peptides from 3 rd round to 6 th round. In both the SCSHP and SCPP biopanning, only 14.90% and 20.91% peptides obtained from TM4 cells in the last in-vitro round were enriched in the testis and comprised 54.67% and 53.84% of the last rounds of in-vivo biopanning ( Figure 2). The overall comparison of the unique peptides found in all four sequencing runs is shown in (Figure 2C). Only 1551 peptides were found common in all the four phage pools sequenced. Combined peptide frequency suggests that few top peptides with a count >10 5 dominated the selected phage pool from the 3 rd and 6 th rounds of both the biopanning experiments ( Figure 1C and D). Only three top peptides were found with a count of >10 5 in the 6 th round of both panning experiments. GSWNTFRAQPTI and YSLRLTSVTAPT are the topmost peptides in SCSHP and SCPP biopanning experiments, respectively, and TGSAKFLQRDTH is the on the 3 rd position in both experiments ( Figure 3). Peptides GSWNTFRAQPTI and YSLRLTSVTAPT were selected for further validation and named SCHP1 and SCHP2, respectively. The top 10 peptides from each of the phage pools sequenced are shown in Table 2. with their respective frequency. Biopanning Data Bank (BDB) [36] search was performed to know whether any of SCHP1 and SCHP2 were previously reported in the literature. SCHP1 was previously reported as a gallium-binding peptide [37], but SCHP2 was not reported anywhere in the literature. A peptide scan was done on SAROTuP [38], a suite of tools for finding potential target-unrelated peptides from phage display data, and we could not find any target-unrelated peptide motifs for both SCHP1 and SCHP2.

The positional abundance of amino acids
The positional amino acid abundance of common 1551 differed from the top 100 common peptides; again, it was different for aligned and non-aligned peptide sequences. In common, 1551 peptides Ser, Pro, Ala, Thr, Arg, and Lys were found to be enriched at the top position ( Figure 4C). However, when aligned, Pro was found to be highly enriched at a specific position ( Figure 4D). Non-aligned top 100 common sequences indicated an enrichment of Ser similar to top 1551 peptides, but other amino acids such as Ala, Pro, Arg, Thr, and Lys were enriched at a few positions ( Figure 4A). Aligned top 100 common sequences showed the highest enrichment of Ser at a specific position, followed by Thr at two positions ( Figure 4B).

Motif discovery and seqlogo
Motif discovery analysis of common 1551 peptide sequences using STREME (Sensitive, Thorough, Rapid, Enriched Motif Elicitation) [39] (https://doi.org/10.1093/bioinformatics/btab203) resulted in six enriched ungapped motifs with p-value <0.05. GSAK motif is enriched in 203 (13.1%) sequences followed by FRAQPTI, YSLRLT,     Figure 3). Though the E-value for these two alignments is <0.1, these motifs are not 100% similar to their hits, indicating the novelty of the discovered motifs. Seqlogo analysis of common 1551 peptides with TBtool ( Figure 5G) revealed GSWNTFRAQ and PTI as enriched domains, which is the actual sequence of the SCHP1 peptide.   . Positional amino acid abundance was calculated with Bioedit software, and heat maps were generated using TB tools.

Homing of SCHP1 and SCHP2 phage to TM4 mouse Sertoli cells
M13 bacteriophages displaying SCHP1/SCHP2 peptides were amplified in E. coli ER2738, and the sequence was confirmed with Sanger sequencing. SCHP1/SCHP2 phages were incubated for 1 h with the TM4 cells, and then the cells were fixed with 4% paraformaldehyde. The homing potential of the SCHP1/SCHP2 phages was checked with anti M13 antibody and detected with Alexa Fluor 594 labelled secondary antibody. Empty phage (w/o 36 bp insert) was used as a control. Confocal image analysis of these cells showed the internalisation of SCHP1 and SSCHP2, displaying phages but not empty phages ( Figure 6). This confirmed the homing potential of the SCHP1 and SCHP2 phages to TM4 cells.

Specific homing of SCHP1 and SCHP2 peptides to TM4 mouse Sertoli cells
uptake of FITC-Ahx-SCHP1 and FITC-Ahx-SCHP1 by TM4 and HEK293 cells were studied with confocal microscopy and quantitatively by flow cytometry. Confocal micrographs of the TM4 and HEK293 show that the peptides SCHP1 and SCHP2 can bind to TM4 cells but not HEK293 cells ( Figure 7A and B). SCHP1 and SCHP2 uptake by Human Coronary Artery Smooth Muscles Cells (HCASMCs) is also lower compared with TM4 cells (Supplementary Figure 4A), and of  scrambled versions of the SCHP1 and SCHP2 peptides by TM4 cells (Supplementary Figure 4B), as evident from the confocal micrographs. As per the quantitation by flow cytometry, the uptake of SCHP1 and SCHP2 by TM4 cells was significantly higher than control TM4 cells at all three concentrations tested. Further, both the peptide's uptake was increased significantly at 200 µM compared to 50 µM and 100 µM. SCHP1 uptake by HEK293 cells was significantly lower than TM4 cells at all three concentrations, but there was no significant difference compared to control HEK293 cells. SCHP2 uptake by TM4 cells was also significantly higher than HEK293 cells at 100 µM and 200 µM concentrations (Figure 7 C and D). The results indicate that the SCHP1 peptide can efficiently target mouse Sertoli cells (TM4) even at lower concentrations, but higher concentrations of SCHP2 are needed for targeting significantly compared with other cell lines. The exact mechanism of SCHP1 and SCHP2 uptake  by TM4 cells is not yet studied, but the literature suggests that it could be through a specific kind of molecular signature present on the surface of the cells [41,42].

Cytotoxicity of the SCHP1 and SCHP2 peptides
Cytotoxicity of SCHP1 and SCHP2 was analysed using MTT cell viability assay at two-fold increasing concentrations. SCHP1 and SCHP2 peptides treated cell was found to be 80% viable at 1280 µM, the highest concentration tested (Figure 8). These results show that the peptides are not cytotoxic and safe for use as cellular-level drug delivery carrier.

Circular dichroism spectroscopy analysis for secondary structure
The secondary structure formation of the SCHP1 and SCHP2 in the environment with different polarities was analysed with Circular Dichroism spectroscopy. Higher percentage of TFE (Trifluroethanol) Figure 9. Circular dichroism spectrum of the synthesised SCHP1 (a) and SCHP2 (B). The peptide solution (0.5 mg/ml) was prepared using 10%, 25%, 50%, 75%, and 90% of Tfe in water. Spectra were collected every 2 nm from 260 to 190 nm. The CD spectra are recorded as CD (mdeg) and represented as Molar ellipticity. mimics membrane bilayer properties by facilitating intramolecular hydrogen bonding [43]. To determine the secondary structure of the SCHP and SCHP2 using CD, the peptides were dissolved in different concentrations of TFE in water. Secondary structures of the SCHP1 ( Figure 9A) and SCHP2 ( Figure 9B) determined using Reed's reference indicates, at the higher concentration of TFE, there is a decrease in the turns and random structure and an increase in the helix structures (Supplementary Table 3), which indicated that the peptide may form more specific helix structures when present around Sertoli cell membrane which is similar to a higher percentage of TFE.

Plasma stability of the SCHPs
Plasma stability of SCHP1/SCHP2 unlabelled, FITC labelled, and Cy5.5 labelled peptides investigated with RP-HPLC. The unlabelled SCHPs were not stable even for 30 min in 25% plasma. FITC labelled SCHP1 and SCHP2 were 50% stable up to 2hand 4 h (Figure 10), respectively. However, Cy5.5 labelled SCHP1 and SCHP2 got rapidly degraded in first 2 h and 50% stable up to 1hand 2 h, respectively and after 2 h the rate of degradation seems to be decreased. Results show that the FITC and Cy5.5 labelled peptides are more stable compared with unlabelled peptides.
1 h, 6 h and 24 h to realise the bio-distribution of the peptides. Total Radiant Efficiency [p/s]/[µW/cm 2 ] in the testis is higher compared to other organs except for the kidney and liver in SCHP1 at all the three-time points, SCHP2 uptake is higher in lungs also along with liver and kidney at 1 h which eventually decreases making testis an organ with higher uptake at 6 h and 24 h time point. In contrast, free Cy5.5 dye showed the highest uptake in the kidney, liver, and lungs at all three-time points ( Figure 11A). Statistical analysis of the Avg Radiant Efficiency [p/s/cm 2 /sr]/[µW/cm 2 ] showed that SCHP1 has significantly higher uptake in the testis compared to heart, brain, and epididymis at 6 h time point and higher uptake in the liver is not significant ( Figure 11B). Accumulation of SCHP2 in testis is significantly higher compared to the heart, spleen and brain at 24 h time point whereas uptake in lungs and kidney is not significantly higher ( Figure 11C). However, in the case of free Cy5.5 dye, there was no significant uptake in the testis at all the time points and uptake by lungs was significantly higher compared to the testis at 1 hand 24 h time points, uptake in the liver was also significantly higher at all the three-time points ( Figure 11D). The uptake of the Cy5.5 labelled SCHP1/SCHP2 is found in the organs other than testis may be because of the degradation of the peptide, as the peptide is not stable till 6 h and 24 h. The results of in-vivo bio-distribution suggest that Cy5.5 tagged SCHP1 and SCHP2 have a different pattern of bio-distribution compared to free Cy5.5 dye, distribution of SCHP1, SCHP2 and Cy5.5 is similar in organs involved in the elimination of the drug from the body such as liver, kidney and lungs [44], but compared to other organs uptake of SCHPs is higher in testis which indicated its targeting efficacy towards testis. When SCHP1/SCHP2 were added to the tissue sections of the testis and liver, higher binding of the SCHP1 was observed in testis compared with the liver (Supplementary Figure 5). However, the binding of the SCHP2 was equal to the testis and liver sections (Supplementary Figure 6). The 2X-confocal micrographs of the testis suggest the binding of the peptide on the surface of the Sertoli cells in the seminiferous tubules ( Supplementary Figures 5 and 6).
The results of the in vivo targeting studies suggest SCHP1 as a more promising target over SCHP2. Increasing the stability of the peptides by replacement of L-amino acids which are not required for homing, by D-amino acids, may increase the targeting potential of the SCHPs.

Conclusions
Conventional drug delivery to treat testicular pathologies requires high dosing frequency, resulting in adverse side effects and systemic toxicity. These issues can be circumvented by specifically targeting testicular cells with the help of specific ligands. Cell-homing peptides are potential candidates as a targeted drug delivery agents and are in clinical trials for various cancer targeting. This is the first study demonstrating the identification of Sertoli cells and testis homing peptides using a phage display peptide library. SCHP1 (GSWNTFRAQPTI) and SCHP2 (YSLRLTSVTAPT) were the two highly enriched topmost peptides during in-vitro and in-vivo rounds of biopanning. Ser, Pro, Ala, Thr, Arg, and Lys are the highly enriched amino acid residues in all the common peptides found in the last rounds of in-vitro and in-vivo biopanning. Motif discovery analyses highlighted motifs GSAK, YSLRLT and SVTAPT, are present in the top two selected SCHP1 and SCHP2 concluding that the biopanning procedure is performed with perfect stringency to select the most suitable peptides. Confocal microscopy of the SCHP1 and SCHP2 displaying phage and flow cytometry of FITC labelled synthetic peptides reveals that both the peptides can specifically target TM4 mouse Sertoli cells and not HEK293 cells and HCASMCs. Synthetic SCHP1 and SCHP2 are non-toxic at the cellular level, and secondary structure prediction suggests the more and helix structure formation in the environment similar to the cell membrane. In-vivo bio-distribution of intravenously injected Cy5.5 labelled SCHPs shows a different pattern than free Cy5.5 dye, and significant enrichment in the testis suggests the testicular targeting potential of the SCHPs. From the results, the selected SCHP1 and SCHP2 peptides have the selective capacity to target Sertoli cells at the in-vitro stage and testis at the in-vivo level. The SCHPs can specifically accumulate in the testis and can serve as an ideal targeting ligand for smart drug delivery to the testis. The efficacy of the male non-hormonal contraceptive drug, like Adjudin, can be increased while decreasing their side effects on other organs. Antioxidants and drugs for treating male infertility whose site of action is inside the testis can also be targeted using these peptides. Plasma stability suggest the stability issues with the selected SCHPs. Various peptides face the same problems of stability, but, addition of a few D-amino acids to the peptide sequence could solve this issue and increase the potential of SCHPs.