Phage Display Identified Novel Leydig Cell Homing Peptides for Testicular Targeting

Abstract

Conventional drug delivery methods to treat testicular disorders face various challenges, which could be circumvented by using targeted drug delivery. Testicular cell targeting ligands, such as Leydig cell homing peptides, would be an excellent choice to achieve the targeted delivery of drugs to the testis. In this study, Leydig cell homing peptides (LCHPs), LCHP1 and LCHP2, were identified via in vitro, followed by in vivo biopanning of a phage display peptide library and next-generation sequencing. Both of the LCHPs were validated in vitro for their specific Leydig cell and in vivo testis targeting potential. Furthermore, molecular targets of the LCHP1 and LCHP2 were identified using affinity purification mass spectrometry (APMS). The LCHP1 and LCHP2 are able to specifically target Leydig cells of the testis and undergo cell internalization as well as target the testis at the in vivo level, hence providing an opportunity to be utilized as a potential ligand for drug delivery to the testis.

Precision medicine and modern drug delivery strategies are taking the front seat in drug development and delivery. Targeted drug delivery enables the delivery of the drug molecules to the desired site of its action, which reduces the side effects on other organs and tissues, hence increasing the efficacy with the reduced dose. Drug delivery can be categorized into passive and active drug targeting. Passive targeting involves the use of the body’s natural response to the physicochemical characteristics of the drug or the drug delivery system. Whereas active targeting uses the homing potential of the targeting ligands to accumulate maximum drug at the site of action.¹ Small molecules, aptamers, peptides, antibodies, and even cells are being used as ligands for the development of tissue-specific targeted drug delivery.² However, peptides pose few advantages over the other targeting ligands, such as being cost-effective, easy to modify, and less immunogenic and hence well tolerated. In addition, the number of drug molecules conjugated to the peptide and the number of peptide molecules decorated on the surface of the nanoparticles is also easy to control.³ Homing peptides specifically target the receptors present on the cell surface and, hence, provide an opportunity for targeted drug delivery. Homing peptide ligands used for cellular drug delivery are of three different types, i.e., cell homing peptides, cell-penetrating peptides, and cell-penetrating homing peptides, depending upon whether the cargo is delivered inside or outside the cells. The Cell homing penetrating peptide targets the specific receptor present on the surface of the particular cell type and then undergoes receptor-mediated endocytosis to deliver the cargo inside the cell.⁴ The advantages of organ homing and cell-penetrating peptides for the targeted delivery of drugs, nanoparticles, liposomes, genes, and proteins have been reviewed by various researchers in the literature reviews.^2,4−7 Identification of homing peptides can be performed using in-silico approaches and phage display libraries. In phage display, the peptide is displayed on the surface of the bacteriophage and the DNA sequence encoding the peptide is present in the phage’s genome.^8,9 The affinity selection method used for the selection of the homing peptide ligands is called biopanning, wherein phages displaying unique peptides are selected based on their affinity to the desired bait, such as cells, receptors, or organs. Over the past two decades, different organ-homing peptides were identified using the phage display technique. In 1996, Pasqualini and Rouslahti attempted for the first time to identify organ-homing peptides.¹⁰ Peptides homing to the breast, adipose tissue, heart, brain, kidney, skin, lungs, pancreas, prostate, muscles, umbilical veins, and synovium have been identified by different researchers using a phage display technology.¹¹⁻²⁰

Infertility affects 8–12% of couples worldwide and 3.9–16% in India and in 50% of the cases, the primary cause is male factor infertility.^21,22 According to the WHO, infertility is the failure to achieve a spontaneous pregnancy despite one year of practicing regular sexual intercourse without contraceptive use. Male infertility possesses a higher risk of having cardiovascular diseases, hypertension, metabolic syndrome, and various cancers, including testicular cancers and hence associated with higher morbidity and mortality.²³⁻²⁶ Therefore, treatment of male infertility is important not only for the couple to bear a child but also for the overall health of the male partner. Current medical treatment strategies for the treatment of male infertility depending upon the cause include hormonal treatment, dopamine agonists, aromatase inhibitors, selective estrogen receptor modulators (SERMs), and antioxidants.^27,28 Infertility associated with hypogonadotropic hypogonadism (HH) can be treated with Gonadotropin replacement therapy, which involves the administration of human chorionic gonadotropin (hCG), a luteinizing hormone (LH) analog; human menopausal gonadotropin (hMG), which mimics LH and FSH; or purified FSH to induce testosterone production by Leydig cells.²⁹ Gonadotropin production can be increased by the administration of the exogenous GnRH, hence the GnRH therapy is useful in case of hypogonadotropic hypogonadism but not for the idiopathic oligo-astheno-teratozoospermia (OAT).³⁰ Elevated levels of FSH in men with testicular failure do not completely justify the administration of the gonadotropins FSH and LH for the treatment of idiopathic OAT and nonobstructive azoospermia (NOA).²⁷ Targeted intratesticular delivery of the FSH to Sertoli cells and LH to Leydig cells instead of systemic administration may justify the use of gonadotropin therapy and improve the outcomes of idiopathic male infertility. Exogenous testosterone therapy is detrimental to spermatogenesis and does not improve fertility outcomes.³¹ Guidelines on male sexual and reproductive health by the European Association of Urology with a 2021 update on male infertility strongly recommend the no use of testosterone therapy for treating male infertility.³² Because administration of exogenous testosterone causes infertility via feedback inhibition of the hypothalamic-pituitary-testicular (HPT) axis, inhibiting GnRH which results in decreased follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone production leading to a decrease in intratesticular concentration.^33,34 However, spermatogenesis and fertility can be restored to normal by intratesticular administration of testosterone.³⁵ Using a targeted delivery system for the intratesticular delivery of testosterone to the testis can improve the efficacy of the testosterone therapy and may not affect the hypothalamic-pituitary-testicular (HPT) axis adversely. Selective estrogen receptor modulators (SERMs), act as an estrogen antagonist at the hypothalamus and pituitary gland and result in increased LH and FSH secretion, which fuels spermatogenesis via increased testosterone biosynthesis.³⁶ Aromatase is an enzyme responsible for the conversion of testosterone and androstenedione to estradiol and estrone, respectively. Infertile males with normal testosterone levels but impaired T/E ratio because of excessive aromatase activity can be treated with aromatase inhibitors such as letrozole and anastrozole.^37,38 Hyperprolactinemia due to prolactin-secreting pituitary adenoma results in infertility via inhibiting the pulsatile secretion of GnRH. Dopamine agonists like bromocriptine and cabergolin are effective in treating the condition of hyperprolactinemia, whereas few patients show resistance to dopamine agonists.^27,39 Enzymatic antioxidants (superoxide dismutase, catalase, and glutathione 4 peroxidase) and nonenzymatic antioxidants (vitamin E, vitamin C, glutathione, and carnitine) supplementation have a beneficial effect in cases of male infertility because of oxidative stress-related sperm dysfunction.⁴⁰⁻⁴³ The efficacy of the antioxidants can be further improved via testis-targeted delivery to neutralize the intratesticular ROS. Approximately 15% of male infertility cases are linked to male genital tract infections (MGTIs). These infections are mainly caused by sexually transmitted pathogens such as Chlamydia trachomatis, E. coli, and Neisseria gonorrhea, and result in an increase in seminal leukocytes.⁴⁴ Based on the nature of the microorganisms identified, antibiotic treatment is decided. Quinolones (ciprofloxacin and levofloxacin) for bacterial infections, tertacyclines and macrolides for Chlamydia trachomatis and Mycoplasma, and trimethoprim is useful against many relevant pathogens except Pseudomonas, some Enterococci, and some Enterobacteriaceae. Antibiotic treatment has been essential to preserving or restoring normal sperm parameters in urogenital infections, but some of them have toxic effects on sperms. This side effect has not yet been directly shown in humans by randomized clinical trials, but there is data concerning testicular and/or sperm toxicity for some antibiotics in rats or mice.⁴⁵ Glucocorticoids such as prednisolone and cyclosporine have been considered for the treatment of antisperm antibodies to reduce inflammatory response but resulted in no significant improvement in fertility and side effects, hence not recommended.^29,46 The targeted drug delivery to the testes could also prove beneficial for the treatment of chronic testicular pain and testicular infection.

Current therapeutic options for the treatment of testicular disorders fall short, because of systemic toxicities. Researchers have tried targeted therapy for the treatment of testicular disorders using anatomical, ultrasound, and ligand-based strategies, but the field remains relatively unexplored. Anatomical targeting of the testis via intra-arterial injection of MSCs for the treatment of torsion-induced testicular injury and repair of germinal cells was useful and the same strategy was efficient in the delivery of nanoparticles to the testis.⁴⁷⁻⁴⁹ Likewise, ultrasound-targeted microbubble destruction (UTMD) has been described as a safe method for the targeted drug delivery of proteins to testis in the rat model.⁵⁰ Testis-specific targeting was achieved via FSH mutant protein 22 amino acid sequence of rat occluding and adjudin both potential male contraceptives.^51,52 In another in vitro study, the FSH peptide-conjugated SOD nanoparticles improved the efficacy of the SOD in the H₂O₂-induced oxidative stress on TM4 cells.⁵³ In a recent study, efficient targeted delivery of isomiR-124a loaded exomes to testis was achieved using FSH (follicle-stimulating hormone), a testis targeting peptide to regulate the expression of sex-determining gene dmrt1 in the testis via isomiR-124.⁵⁴ Recently, our group has identified Sertoli cell homing peptides using a phage display library for targeted drug delivery to testis.⁵⁵ Germ cells, Sertoli cells, Leydig cells, and endothelial cells of the testicular microcapillaries are the potential cellular targets inside the testis, which could be targeted for the testis-specific drug delivery and treatment of different testicular disorders. Male gametes and androgen production are two important functions performed by the testis. These two functions occur in the two structurally distinct compartments, i.e. seminiferous tubules and interstitial compartment of the testis.⁵⁶ Adult Leydig cells are the most essential cells in the interstitial compartment and are involved in testosterone production, which is essential for the maintenance of normal spermatogenesis.⁵⁷ In this present study, efforts are made to identify the Leydig cell homing peptides (LCHPs) using a phage display library. The LCHPs could be used as a targeting moiety for designing targeted drug delivery to the testis.

Results

Phage Display Library Screening for Identification of Leydig Cell Homing Peptides (LCHPs)

In vitro biopanning on TM3 (mouse Leydig cell line) was performed to select Leydig cell surface homing peptides (LCSHPs) and Leydig cell-penetrating peptides (LCPPs). Phages obtained after the third cycle of the in vitro biopanning were used for in vivo biopanning with the Balb/C mouse model. Three rounds of the in vivo biopanning procedure were performed with Balb/C mouse to select testis-homing peptides from the LCSHPs and LCPPs (Figure 1).

Figure 1.

Scheme of the biopanning experiment performed for the screening of the Leydig cell homing peptides (LCHPs). (a) In vitro panning (three rounds) on TM3 (mouse Leydig cell line) to select Leydig cell surface homing peptides (LCSHPs) and Leydig cell-penetrating peptides (LCPPs). (b) In vivo panning (three rounds) in a mouse model using restricted LCSHPs and LCPPs obtained from the third round of in vitro panning.

Titration of the phage pool after each round of biopanning suggested the enrichment of the phages in the third round as compared to the first round of in vitro biopanning and the sixth round compared to the fourth round of in vivo biopanning for both the LCSHPs and LCPPs experiments (Figure S3a). The increase in the titer suggested the successful enrichment of LCSHPs and LCPPs. Next-generation Illumina sequencing of the phage pool of round 3, i.e. of in vitro, and round 6, i.e., of in vivo biopanning experiments revealed the unique peptide sequences. Peptide diversity in the sixth (in vivo) round of both LCHSPs and LCPPs was found to be reduced compared with that in the third round (in vitro) (Figure S3b). Identification of the peptide sequences from selected phage pools was performed with next-generation sequencing (NGS). The phage pools from the third round and sixth round were pooled together to make one sample of LCSHPs and LCPPs each (Table S6). Approximately equal number of proper reads were obtained after NGS (Table S7). The fraction of peptides with frequencies >10⁴ was very few, but their combined frequencies were high in the third round and higher in the sixth round of both LCSHPs and LCPPs (Figure S3c). A comparison of the unique peptides identified in the third and sixth rounds of LCSHPs and LCPPs is shown as a Venn diagram in Figure 2. Only 323 unique peptides from the third round were enriched in the sixth round of LCSHPs and 354 unique peptides were enriched for LCPPs from the third to sixth round (Figure 2a,b). A comparison of unique peptides found in the third and sixth rounds of both LCSHPs and LCPPs biopanning resulted in only 127 common unique peptides, which are called Leydig cell homing peptides (LCHPs) (Figure 2c).

Figure 2.

Summary of unique peptide identified from the 3rd round and 6th round phage pools of LCSHPs (a) and LCPPs (b) and comparison between all four rounds (c). A comparison of Leydig cell homing peptides (LCHPs) with Sertoli cell homing peptides (SCHPs) from the previous study (d and e). Scatter plot of the 127 LCHPs showing their frequency in 3rd and 6th rounds of LCSHPs and LCPPs pools (f).

The scatterplot in Figure 2f represents the actual frequency of the 127 LCHPs in all four rounds sequenced, wherein LCHP1 (HHGANSLGLVQS) and LCHP2 (YALGRPSLQGPN), the two peptides with the highest frequencies, were selected for further characterization. The top ten LCHPs based on their enrichment from the third round to the sixth round are enlisted in Table 1. LCHP1 and LCHP2 are the only two peptides with an enrichment of over 10%. When all LCSHPs and LCPPs were compared with all of the Sertoli cell homing peptides (SCSHPs) and Sertoli cell-penetrating peptides (SCPPs) from our previous study, 103 common peptides were found that can target the testis and have both Leydig and Sertoli cell homing potential inside the testis (Figure 2d), whereas the comparison between selected LCHPs and selected SCHPs resulted in only one common peptide with the amino acid sequence of FKQDAWEAVDIR (Figure 2e).

Table 1. Top 10 Selected Leydig Cell Homing Peptides (LCHPs) with Their Frequency in 3rd and 6th Rounds of Bio-Panning Performed for the Identification of Leydig Cell Surface Homing Peptides (LCSHPs) and Leydig Cell Penetrating Peptides (LCPPs) and Their Percent Enrichment from the 3rd Round to the 6th Round.

LCHPs	R3 LCSHPs frequency	R6 LCSHPs frequency	LCSHPs enrichment (%)	R3 LCPPs frequency	R6 LCPPs frequency	LCPPs enrichment (%)
HHGANSLGLVQS	25,805 (21.150%)	117,522 (87.368%)	66.218	220 (0.271%)	60,058 (36.938%)	36.667
YALGRPSLQGPN	2125 (1.742%)	2657 (1.975%)	0.234	585 (0.720%)	19,786 (12.170%)	11.448
NQCKECFIRAGD	1093 (0.896%)	1642 (1.220%)	0.325	626 (0.771%)	13,249 (8.1497%)	7.377
NESGITRIALQD	7 (0.005%)	62 (0.047%)	0.040	33 (0.040%)	598 (0.368%)	0.327
HHGANSLGLVRS	37 (0.030%)	203 (0.150%)	0.121	2 (0.002%)	152 (0.093%)	0.091
HHGANSLGLMQS	30 (0.024%)	135 (0.100%)	0.076	1 (0.001%)	78 (0.0480%)	0.047
HHGENSLGLVQS	23 (0.019%)	100 (0.074%)	0.055	1 (0.001%)	67 (0.041%)	0.040
HHGADSLGLVQS	21 (0.018%)	93 (0.070%)	0.052	1 (0.001%)	60 (0.037%)	0.036
YALGRPSLQGPS	3 (0.002%)	11 (0.009%)	0.006	3 (0.003%)	36 (0.022%)	0.018
NQRKECFIRAGD	3 (0.002%)	4 (0.002%)	0.001	2 (0.002%)	28 (0.017%)	0.015

Motif Discovery and In-Silico Analysis of the LCHPs

To identify the enriched motifs in the selected 127 LCHPs, in silico analysis was performed on the STREME (Sensitive, Thorough, Rapid, Enriched Motif Elicitation) server.⁵⁸ GRPS, YAL, GPN, and KQD are the four identified motifs shown in Figure S4a–d. The summary of the P value, E value, and number of sites (peptide sequences) in which the sequence was found is given as a table in Figure S4e. GRPS, YAL, and GPN motifs are the actual sequences present in the LCHP2, which shows the probable involvement of these motifs in the homing of the LCHP2 to the Leydig cells.

The protein blast of LCHP1 using UniProt resulted in three hits: ferrous iron transporter B, porin, and SPATA31 domain-containing protein. LCHP2 showed sequence similarity with DNA-protecting protein DprA, uncharacterized protein, transcription factor (Sin3), sin3 family corepressor, histone deacetylase interacting domain-containing protein, cation-transporting P-type ATPase C, AAA+ ATPase domain-containing protein, cytochrome P450, fibronectin type-III domain-containing protein, ABC transporter permease subunit, ranBP2-type zinc finger protein At1g67325-like isoform X1, and ranBP2-type domain-containing protein. Details of the blast results are shown in Figure S5.

In Vitro Characterization for the Homing Potential of LCHP1 and LCHP2

Qualitative and quantitative analysis of the TM3 (mouse Leydig cells) homing potential of FITC-tagged LCHP1 and LCHP2 was shown by using confocal microscopy and flow cytometry. TM4 (mouse Sertoli cells) and H9C2 (rat embryonic cardiomyocytes) were used as control/nonspecific cells. TM3 cells showed higher uptake of LCHP1 and LCHP2 compared with TM4 and H9C2 cells in the confocal micrographs, as shown in Figure 3a–c, which suggest the specificity of LCHP1 and LCHP2 toward Leydig cells.

Figure 3.

In vitro uptake of LCHP1 and LCHP2 by TM3, TM4, and H9C2 cells. Confocal micrographs of LCHP1 and LCHP2 treated TM3 (a), TM4 (b), and H9C2 (c) cells. (d) Histograms of the quantitative uptake measurement LCHP1 and LCHP2 uptake at 50, 100, and 200 μM by flow cytometry. e. Statistical analyses of the quantitative uptake of LCHP1 and LCHP2 by TM3, TM4, and H9C2 cells. All data represent the mean ± SD; n = 3; * represents p < 0.05, ** represents p < 0.001, and **** represents p < 0.0001 for two-way ANOVA, followed by Tukey’s multiple comparisons test.

Quantitative analysis of uptake of LCHP1 and LCHP2 by TM3, TM4, and H9C2 cells was performed by using flow cytometry (Figure 3d,e). LCHP1 showed significantly higher uptake in TM3 compared with TM4 and H9C2 cells at 100 and 200 μM concentrations. Whereas, LCHP2 showed significantly higher uptake in TM3 than TM4 at 50, 100, and 200 μM concentrations. The TM3 equiv uptake of LCHP2 by H9C2 cells at 200 μM and higher uptake at 50 and 100 μM, could be because of autofluorescence of the cells because the unstained H9C2 also showed higher fluorescence intensity. Also, the in vivo biodistribution of the LCHP2 did not show significant uptake in the heart, which suggests the higher uptake by H9C2, which are rat cardiomyocytes, may not be the concern for targeting Leydig cells in the in vivo system.

In Vivo Biodistribution and Testis Homing Potential of LCHP1 and LCHP2

In vivo biodistribution of the Cy5.5 tagged LCHP1, LCHP2, and a control peptide CTP was performed by the use of an in vivo imaging technique, wherein 20 μg of each peptide was injected into the mice intravenously and the animals were sacrificed after 1, 6, and 24 h of circulation. Representative images of the organs at 1, 6, and 24 h of the LCHP1-Cy5.5, LCHP2-Cy5.5, and CTP are shown in Figure 4a.

Figure 4.

In vivo biodistribution of LCHP1-Cy5.5, LCHP2-Cy5.5, and CTP-Cy5.5 using IVIS Lumina in vivo imaging system. (a) Representative organ images of LCHP1-Cy5.5, LCHP2-Cy5.5, and CTP-Cy5.5 at 1, 6, and 24 h time point. (b) Statistical analyses of average radiant efficiency comparison between organs. (c) Statistical analysis of the percent peptide distribution in organs and comparison between LCHP1-Cy5.5, LCHP2-Cy5.5, and CTP-Cy5.5. All data represents the mean ± SD; n = 3; * represents p < 0.05, ** represents p < 0.001, and **** represents p < 0.0001 for two-way ANOVA, followed by Tukey’s multiple comparisons test.

The statistical analyses of the average radiant efficiency due to Cy5.5 dye conjugated to the peptides in the testis and other vital organs are shown in Figure 4b. Testis showed a significantly higher accumulation of LCHP1-Cy5.5 compared with the heart at all three time points, i.e., 1, 6, and 24 h. The accumulation of LCHP1-Cy5.5 was significantly higher in the testis compared with the spleen at 1 h and compared with the spleen, brain, and epididymis at 6 h time points. LCHP2-Cy5.5 showed better uptake in the testis than LCHP1-Cy5.5 with a significantly higher accumulation compared with heart and spleen at 1, 6, and 24 h. LCHP2-Cy5.5 also showed significantly higher accumulation in the testis compared with the brain, epididymis, and seminal vesicles at 6 and 24 h time points. The signal because of LCHP1-Cy5.5 and LCHP2-Cy5.5 rapidly decreased with time in the liver and kidneys. Whereas, the CTP-Cy5.5 distributions were more uniform in all the organs and there was no significant difference in the accumulation of peptide in all analyzed organs except the liver and kidneys where the uptake was significantly higher in the latter two compared with the testis.

Figure 4c shows the comparative percent peptide distribution in LCHP1-Cy5.5, LCHP2-Cy5.5, and CTP-Cy5.5. At a 1 h time point, the percent distribution of the Cy5.5 labeled LCHP1 is higher in all organs except the liver and kidneys compared to CTP, but the uptake in the testis is significantly higher. Also, LCHP2-Cy5.5 was higher in all organs except only the liver compared with CTP, whereas the percent distribution of CTP was significantly higher in the liver and lower in all other organs compared with both LCHP1 and LCHP2. At a 6 h time point, the percent distribution of the LCHP1-Cy5.5 was higher compared with CTP in all organs and lower in the liver and kidneys, but the distribution in testis, brain, epididymis, and seminal vesicles was significantly higher and the difference in the testis was more significant.

LCHP1-Cy5.5 distributions were higher compared with those of CTP in all organs and significantly lower in the liver and kidneys, but the difference is significant only in the testis, whereas the distribution of CTP was significantly higher in the liver and kidneys compared with LCHP1 and LCHP2. At the 24 h time point, LCHP1-Cy5.5 and LCHP2-Cy5.5 distribution compared with CTP was higher in all the organs but significantly lower in the liver and kidneys.

As expected, a higher distribution of the peptides was observed in the liver and kidneys as the intravenously injected peptides accumulate and leave the body via these organs.^59,60 LCHP distribution in the testis decreases with the time after 6 h, which could be because of the degradation of the peptide and fluorescent dye conjugate as the signal is from the conjugated dye. The best distribution of the LCHP1 and LCHP2 was observed at a 6 h time point, and LCHP2 has a better testis targeting potential compared to LCHP1.

Safety and Cytotoxicity Assessment of LCHPs

To assess the in vitro safety of LCHPs, an MTT assay was performed wherein the TM3 cells were treated with two-fold increasing concentrations of both LCHP1 and LCHP2 for 48 h. The TM3 viability was over 80% at the highest tested concentration of 1280 μM for both peptides. Moreover, LCHP1 and LCHP2 did not show any differences in cell viability at the tested concentrations (Figure S6). The results show that the peptides LCHP1 and LCHP2 are safe for TM3 cells and do not cause toxicity.

Plasma Stability Assessment of the LCHPs

The plasma stability of peptides was assessed at different time points by incubation of nontagged, FITC-tagged, and Cy5.5 tagged LCHP1/LCHP2 in the 25% mouse plasma (Figure 5). It is noteworthy that the FITC and Cy5.5 tagged peptides show higher stability than the unlabeled ones. And, the FITC-tagged peptides are more stable than Cy5.5-labeled ones. The tagged versions of the LCHP2 were stable for a longer time compared to LCHP1.

Figure 5.

Plasma stability of unlabeled, FITC labeled, and Cy5.5 labeled LCHP1 (a) and LCHP2 (b) at different time points. The percent stable peptide at a given time point was calculated considering 0 min’ concentration as 100%.

Secondary Structure Analysis of LCHPs

The secondary structure of the LCHP1 and LCHP2 was analyzed by CD spectroscopy in different concentrations of TFE (Figure 6), which mimics the cell membrane-like environment.⁶¹ With the increase in the TFE concentration, a decrease in the random structures was observed for both LCHP1 and LCHP2. The helix structures appeared at 50% TFE and increased with higher TFE concentration. Higher beta structures were observed at 25% TFE and a further increase in TFE concentration did not affect the beta structure formation. If compared between LCHP1 and LCHP2, LCHP2 displays fewer random structures and more beta structures (Table S8a and S8b).

Figure 6.

Circular dichroism spectroscopy of LCHP1 (a) and LCHP2 (b). The peptides were dissolved at 0.5 mg/mL concentration in 10, 25, 50, 75, and 90% TFE in water. The spectra in CD (mdeg) were recorded at every 2 nm from 260 to 190 nm on a Jasco J-810 spectropolarimeter using a 1 mm cuvette.

Molecular Target Identification of LCHPs from TM3 and Testis Membrane Proteins

To purify the molecular targets of LCHP1 and LCHP2 on the surface of Leydig cells, affinity chromatography was performed with membrane proteins of TM3 cells and testis tissue. After the affinity purification, the affinity eluates were run on SDS-PAGE (Figure 7). In eluate from LCHP1 and LCHP2 affinity chromatography, a specific band around 27 kDa (lanes 2 and 5) and 85 kDa (lanes 3 and 6), respectively, was observed in SDS-PAGE. No such specific bands were observed in the eluate from no peptide control (lanes 4 and 7) but a few bands around 70 kDa were present in all the samples, which could be the proteins binding nonspecifically to the agarose medium. The specific bands obtained from LCHP1 and LCHP2 affinity chromatography were subjected to gel trypsin digestion, followed by mass spectrometry for protein identification. The list of all the proteins identified as potential targets for LCHP1 and LCHP2 is given in Table S9a and Table S9b, respectively.

Figure 7.

Silver stained SDS-PAGE of the affinity eluate. Lanes 1 to 7 represent 1. protein molecular weight marker, 2. LCHP1 affinity eluate from TM3 membrane proteins, 3. LCHP2 affinity eluate from TM3 membrane proteins, 4. No-peptide affinity eluate from TM3 membrane proteins, 5. LCHP1 affinity eluate from testis membrane proteins, 6. LCHP2 affinity eluate from testis membrane proteins, and 7. No-peptide affinity eluate from testis membrane proteins.

AP-MS data suggest the Prdx2 protein (Q5M9N9) and/or peroxiredoxin-1 (P35700) as potential targets of the LCHP1 peptide in Leydig cells and testis. Protein expression levels of PRDX1 and PRDX2 are reported to be the highest in the Leydig cells. Another protein PRDX6 was also reported to be high in Leydig cells and Sertoli cells equally.⁶² Membrane-bound presence of peroxiredoxins is also reported in a few studies.⁶³⁻⁶⁵

LCHP2 might target spermatogenesis-associated protein 20 (Q80YT5) or junction plakoglobin (Q02257) proteins expressed by Leydig cells and testis. Spermatogenesis-associated protein 20 (Spata20) also known as sperm-specific protein 411 (Ssp411) is a member of the spermatid-specific thioredoxin family.⁶⁶ The human protein atlas data show the medium-level expression of Spata20 in Leydig cells after elongated spermatids. LCHP2 may target Spata20 present on the Leydig cell membrane. The other protein junctional plakoglobin, also known as plakoglobin or gamma catenin, is present in adherens junctions and desmosomes.⁶⁷ According to the literature, the plakoglobin is expressed at the basal seminiferous tubules, concentrated at Sertoli cell–Sertoli cell junctions and not at the Sertoli cell-germ cell junctions pattern in testis, which is similar to alpha-catenin.⁶⁸ The presence and role of Junctional plakoglobin in adult Leydig cells are not well demonstrated in the literature, although it could be present in Leydig cells and more accessible to the LCHP2 peptides because of its presence in the interstitial compartment.

Discussion and Conclusions

Current treatment strategies to treat testicular disorders are hindered by the adverse side effects of systemically administered drugs, high dosing frequency, inability to achieve sufficiently high intratesticular concentrations for therapeutic activity, and failure of large molecules to cross the blood–testis barrier. However, these issues can be addressed by the use of targeted drug delivery to the testis. The area of testicular targeting has not been much explored and is limited mainly to anatomical targeting of the testis. But, with the advancements in drug targeting strategies, the treatment modalities can be further improved. Leydig cell-targeted delivery of the drugs can be explored for the treatment of male infertility and other testicular disorders. As a first step toward developing Leydig cell-targeted drug delivery to the testis, we have identified Leydig cell homing peptides using the phage display peptide library.

The screening of the peptides was performed by using a phage display library, which is a well-known method for the identification of homing peptides. However, the most stringent approach was used for the screening of LCHPs. Here the biopanning was performed in vitro for LCSHPs and LCPPs followed by in vivo in a mouse model, which ensured the selection of the peptides that can target Leydig cells and get internalized as well as able to target testis at the in vivo level. Next-generation sequencing-based identification could provide the maximum coverage of the LCHP frequency and facilitate the selection of the best candidate peptides, i.e., LCHP1 and LCHP2. Further, In vitro uptake studies of the labeled LCHP1 and LCHP2 showed their specific homing capabilities toward TM3 (mouse Leydig cells). In vivo biodistribution shows the testis targeting potential of the selected LCHPs. The unlabeled LCHP1 and LCHP2 are not stable enough, and further efforts are required to increase the stability of the same. Conjugation of the drug molecules or drug nanoparticles to these peptides may also increase the life span of the peptides in plasma, which remains to be explored. Information about the potential molecular targets of LCHPs could be useful in predicting the suitability of these peptides for human use.

We believe that LCHP1 and LCHP2 are excellent ligands for targeting Leydig cells and can be further explored for the development of targeted drug delivery systems. Using these homing peptides as targeting ligands for the delivery of any drugs to the testis could increase the therapeutic efficacy while reducing the side effects on the nontargeted organs. The study also demonstrates the identification of the molecular targets of the selected LCHPs. However, further validation may provide some insights into the mechanism of homing of these peptides into Leydig cells. These LCHPs can be conjugated with drugs for the treatment of various testicular disorders, including male infertility, testicular cancer, testicular oxidative stress, etc.

In conclusion, LCHP1 and LCHP2, peptides homing to Leydig cells and able to target testis, were identified using phage display. The peptides were validated for their homing capabilities using in vitro and in vivo studies. However, there is a need to study the testis-specific drug delivery potential of LCHP1 and LCHP2. Furthermore, efforts are made to identify the molecular targets LCHP1 and LCHP2, but further validation of the targets is required.

Experimental Section

Phage Display Library

Ph.D.-12, a 12-mer Phage display library from New England Biolabs, was used in this study. The library comprised approximately 1 × 10⁹ unique sequences.

Cell Culture

TM3 (mouse Leydig cells) (ATCC CRL-1714) and TM4 (mouse Sertoli cells) (ATCC CRL-1715) were purchased from an American-type culture collection, and H9C2 was purchased from the National Centre for Cell Sciences (NCCS) Pune. Dulbecco’s modified eagle medium (DMEM) nutrient mixture F-12 supplemented with 2.5% fetal bovine serum and 5% horse serum was used to culture TM3 and TM4 cells, and DMEM nutrient mixture F-12 (Ham) (1:1) supplemented with 10% fetal bovine serum for H9C2 cells. All the cells i.e., TM3, TM4, and H9C2 were maintained in culture at 37 °C with 5% CO₂.

Animal Use and Care in the Study

All the procedures were performed on 6–8 week adult Balb/C male mice, which were approved by ICMR-National Institute for Research in Reproductive and Child Health Mumbai’s Institutional Animal Ethics Committee (IAEC-03/20).

In Vitro Biopanning

In vitro biopanning was performed on TM3 (mouse Leydig cells) as described earlier in Jirwankar and Dighe 2023 and McGuire et al.^55,69 Briefly, 0.5 × 10⁶ cells/well were seeded before 48 h of biopanning in 6-well plates. On the day of biopanning, the spent medium was replaced with DMEM/F12 without serum for receptor clearance and cells were incubated for 1 h. Then cells were treated for 1 h with a biopanning solution containing 1 × 10¹¹ phages of PhD-12 library and protease inhibitor in 0.1% BSA (bovine serum albumin) containing PBS (phosphate buffered saline). Unbound phages were washed with PBS containing 0.1% BSA four times for 5 min each. After the washing of unbound phages, the Leydig cell surface homing phages (LCSHPs) were eluted with 1 mL of 0.1 M HCl-Glycine, pH 2.2 + 0.9% NaCl. Again, the cells were washed twice using 0.1% BSA containing PBS to discard any cell surface homing phages. Further, 1 mL of 30 mM Tris–HCl, pH 8.0, a hypotonic solution, was added to wells, and cells were incubated for 30 min on ice. The cells were frozen at −20 °C overnight, and the next day, Leydig cell-penetrating phages (LCPPs) were recovered from the disrupted cells (Figure 1).

In Vivo Biopanning

In vivo biopanning was performed in Balb/C male mice wherein, amplified Leydig cell surface homing phage pool and Leydig cell-penetrating phage pool from the last round of in vitro biopanning (3rd round) were used to perform the first round of in vivo biopanning. Briefly, 1 × 10¹¹ phages were injected intravenously into mice, and after 30 min of circulation, mice were sacrificed, and all vital organs were collected and stored at −80 °C. Testis tissue was minced in the protease inhibitor-containing DMEM medium, homogenized, and centrifuged, and the supernatant was collected which contained phages from the testis. The phages recovered from the testis were amplified for the next round of biopanning via infecting Escherichia coli ER2738 cells. Three rounds of in vivo panning were performed for the enrichment of the specific phages^55,70 (Figure 1).

Phage Amplification after Biopanning Rounds

Phage amplification after each round of biopanning was performed by infecting the E. coli ER2738 host cells, as described in the phage display library manufacturer’s manual. The overnight culture of ER2738 was diluted in fresh 50 mL of LB broth, and phages obtained after each round of in vitro and in vivo biopanning were added to it. The culture was allowed to grow for 4.5–5 h at 37 °C. After the incubation was over, E. coli ER2738 were separated with centrifugation for 10 min at 12,000g at 4 °C, and 1/6 volume of 20% PEG/2.5 M NaCl was added to the supernatant to allow phage precipitation overnight at 4 °C. Next day, the precipitated phages were isolated by centrifugation at 12,000g at 4 °C for 15 min, and the pallet was dissolved in TBS (Tris-buffered saline), followed by titration.

Phage Titration after Biopanning Rounds

Phage titration of the phage pool obtained after each round of biopanning (unamplified and amplified) was performed with the qPCR standard curve method (Figure S1) as described earlier in Jirwankar and Dighe⁵⁵ and Peng et al.,⁷¹ wherein 0.1 to 10⁶ fg/μL (2.689 × 10¹ to 2.689 × 10⁸ gc/μL) of M13 ssDNA were used as standards. Phages were precipitated with PEG (Polyethylene glycol)/NaCl and treated with DNase I at 37 °C for 10 min, followed by heat denaturation at 100 °C; to get the phage DNA template for the qPCR. Details of primers, reaction mixture, and reaction conditions are given in the Supporting Information Tables S1 and S2. The reaction was performed on Applied Biosystems’s QuantStudio5 Real-time PCR and analyzed with QuantStudio Design and Analysis software.

Next-Generation Sequencing of Phage Pool

As described earlier in the study of Jirwankar and Dighe,⁵⁵ next-generation Illumina sequencing was performed for the identification of the displaying peptide sequences. Phage DNA (ssDNA) was isolated from the phage pools as described by Green et al.,⁷² and PCR was performed to prepare 77 bp amplicon for Illumina sequencing (Figure S2a); forward primer and reverse primer with barcodes used were picked from Matochko et al.⁷³ Details of primers, reaction mixture and reaction condition used are provided in the Supporting Information Tables S3–S5. The final PCR product of 77bp was run on 2% agarose gel (Figure S2b) and was extracted using a GeneJET gel extraction kit and further submitted for Illumina sequencing at Bioxplore Laboratories, Chennai, India.

Peptide Synthesis

FITC and Cy5.5 tagged LCHP1-Ahx-FITC, LCHP2-Ahx-FITC and {C (Cy5.5)}-LCHP1, {C (Cy5.5)}-LCHP2, and {C (Cy5.5)}-CTP with 99% purity were purchased from Lifetein, LLC, USA. Biological-grade LCHP1 and LCHP2 with >95% purity were purchased from AsianBioChem, Thrissur, Kerala, India.

Confocal Microscopy of the LCHPs with TM3, TM4, and H9C2 Cells

TM3, TM4, and H9C2 cells were seeded overnight onto presterilized coverslips at a density of 0.1 × 10⁶ cells per well. The next day, cells were treated with 200 μM of FITC tagged LCHP1, and LCHP2 for 1 h. After the incubation, cells were washed three times with calcium and magnesium-containing DPBS (Dulbecco’s phosphate buffered saline), fixed with 4% paraformaldehyde, and counter-stained nuclear staining with DAPI, and cytoskeleton staining with rhodamine-phalloidin (Invitrogen Cat #R415). Coverslips were mounted on slides and analyzed by confocal microscopy.

Flow Cytometry of the LCHPs with TM3, TM4, and H9C2 Cells

For the flow cytometry analysis, 0.5 × 10⁶ TM3, TM4, and H9C2 cells per well were seeded in 6-well plates. On the next day, cells were treated with 50, 100, and 200 μM concentrations of FITC-tagged LCHP1 and LCHP2 for 1 h. After the incubation, cells were detached using a 1 mM EDTA (ethylenediaminetetraacetic acid) solution, washed three times with DPBS, and analyzed with the flow cytometer. Data analysis was performed with Floreada software.

MTT Assay

The safety of LCHP1 and LCHP2 was assessed with an MTT assay on TM3 cells. Briefly, 0.05 × 10⁶ cells per well were seeded in 96-well plates and incubated overnight. The cells were treated for 48 h at various concentrations of LCHP1/LCHP2 peptides. The next day, 50 μg of MTT solution was added to each well and incubated for 4 h at 37 °C. To solubilize the formazan crystals, 100 μL of DMSO (dimethyl sulfoxide) was added to each well after removal of the MTT solution, and the plate was incubated at room temperature for 30 min. The plate was read for OD at 570 nm, and the percent cell viability was calculated.

Circular Dichroism Spectroscopy

The peptides LCHP1 and LCHP2 (0.5 mg/mL) were dissolved in 10, 25, 50, 75, and 90% trifluoroethanol (TFE) in water. Circular dichroism (CD) spectroscopy was performed on a Jasco J-810 spectropolarimeter using a 1 mm cuvette. Spectra were collected every 2 nm from 260 to 190 nm. The CD spectra are reported as CD [mdeg], and the secondary structures were determined using Reed’s reference.

Plasma Stability Study

Plasma stability of the LCHP1 and LCHP2 nontagged peptide, FITC tagged, and Cy5.5 tagged peptides were studied in the mouse plasma with HPLC. Peptides were dissolved in 10 μL of DMSO and 1 mL of 25% mouse plasma in DMEM medium and incubated at 37 °C. At 0, 0.25, 0.5, 1, 2, 4, 6, and 8 h time points, 100 μL of the mixture was taken, and plasma proteins were precipitated with the addition of 200 μL of 95% chilled ethanol. The mixture was vortexed and centrifuged at 13,000g for 2 min at 4 °C, and the supernatant was collected. To study the stability of the peptide, 60 μL of it was injected into the C18 column, and RP-HPLC analysis was performed. Mobile phase A of 0.1% TFA in water and phase B 0.1% TFA in acetonitrile in a linear gradient of 25–80, 25–65, and 25–50% of phase B was used for Cy5.5 tagged, FITC tagged, and nontagged peptides, respectively, as a mobile phase for 20 min. UV detection wavelengths were set at 220, 541, and 675 nm for nontagged peptides, FITC tagged, and Cy5.5 tagged peptides, respectively. The stability percentage of the peptide at different time points was determined by considering 0 min peptide concentration as 100% peptide.

In Vivo Biodistribution

In vivo biodistribution was performed on 6 to 8-week-old adult male Balb/C mice. The Cy5.5 tagged peptides were purchased from LifeTein LLC. Mice (n = 3) were injected intravenously with 20 μg of the peptides LCHP1, LCHP2, and CTP (a control peptide) each tagged with Cy5.5 in 100 μL of phosphate buffered saline. Mice were sacrificed at 1, 6, and 24 h after peptide injection, and the testis and other vital organs were imaged using the IVIS lumina III in vivo imaging system. Image analysis was performed by using Living Image software.

Affinity Purification Mass Spectrometry (AP-MS)

Membrane protein isolation for affinity purification was done using a Mem-PER Plus membrane protein extraction kit from Thermo Scientific (cat. no. 89842) and affinity purification was performed using Pierce NHS-activated agarose spin columns (Cat# 26198). Phosphate buffered saline of pH 7.2 containing 50 mM octyl glucoside and 1 mM PMSF (phenylmethylsulfonyl fluoride) was used as wash buffer/binding buffer/coupling buffer/column buffer. Peptide immobilization to NHS-activated agarose was performed overnight at 4 °C using 2 mg/mL peptide in coupling buffer with end-over end mixing. The next day, the columns were washed to remove unbound peptide and the remaining active sites were blocked for 1 h at room temperature with quenching Buffer (1 M Tris pH 7.4). Membrane proteins in membrane solubilization buffer from TM3 cells/testis were diluted in binding buffer (1:1) and added to the column, binding of the proteins to the immobilized peptide on the column was allowed for 2 h at room temperature with end-overend mixing. The columns were washed three times with wash buffer, and then elution was performed in the column buffer containing 1 mg/mL peptide. The eluates were concentrated 5 times using a concentrator with a 3 kDa cutoff and further run on the 12% SDS-PAGE and stained with silver staining. The desired band from stained SDS-PAGE was cut and subjected to in-gel tryptic digestion, followed by peptide extraction and desalting of the peptides. The peptides were run on Thermo Scientific’s Q-Exactive Plus Biopharma-High Resolution Orbitrap mass spectrometer at a sophisticated analytical instrumentation facility (SAIF), IIT Bombay, and protein identification was performed with Proteome discoverer software.

Statistical Analysis

All the statistical analysis was performed on GraphPad Prism version 10.0.1. Two-way ANOVA was performed followed by Tukey’s multiple comparisons test for the analysis of flow cytometry and in vivo biodistribution data.

Glossary

Abbreviations

LCHPs

Leydig cell homing peptides

LCSHPs

Leydig cell surface homing peptides

LCPPs

Leydig cell penetrating peptides

APMS

Affinity purification mass spectrometry

GnRH

gonadotropin-releasing hormone

rec-hCG

human chorionic gonadotropin

hMG

human menopausal gonadotropin

rec-hFSH

follicle-stimulating hormone

rec-hLH

luteinizing hormone

SERMs

selective estrogen receptor modulators

OAT

oligo-astheno-teratozoospermia

NOA

nonobstructive azoospermia

HPT

hypothalamic-pituitary-testicular

FSH

follicle-stimulating hormone

ROS

reactive oxygen species

SOD

superoxide dismutase

MSCs

mesenchymal stem cells

UTMD

ultrasound-targeted microbubble destruction

TFE

trifluoroethanol

Supporting Information Available

. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00330.

• Details of the PCR reaction condition; primer used and reaction conditions for PCR and qPCR in the table formats; summary of NGS reads; details of CD spectroscopy and details of protein identified by mass spectrometry; supporting figures for qPCR titration; details of amplicon preparation for NGS; phage titration results; peptide diversity figure; motif discovery analysis; blast result summary of LCHPs; MTT assay results graph (PDF)

• Common LCHPs and percentage information (XLSX)

Author Contributions

Conceptualization and Methodology: Y.J. and V.D., Formal Analyses: Y.J., Investigation: Y.J., Project Administration: V.D., Writing (Original Draft): Y.J., Writing (Review and Editing): V.D. and Y.J., Supervision: V.D., Funding Acquisition: V.D. and Y.J. S.M. did the mass spectrometry and analysis, A.N. performed the in vitro peptide uptake studies under Y.J.’s supervision.

This work was supported by the Department of Health Research, Government of India [grant no: R.11013/39/2021-GIA/HR].

The authors declare no competing financial interest.