Drug delivery to the testis: current status and potential pathways for the development of novel therapeutics
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- Snow-Lisy, D.C., Samplaski, M.K., Labhasetwar, V. et al. Drug Deliv. and Transl. Res. (2011) 1: 351. doi:10.1007/s13346-011-0039-x
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Nanotechnology has been increasingly utilized for the targeting and delivery of novel therapeutic agents to different tissues and cell types. The current therapeutic options for testicular disorders fall short in many instances due to difficulty traversing the blood–testis barrier, systemic toxicities, and complicated dosing regiments. For testicular tissue, potential targeting can be obtained either via anatomic methods or specific ligands such as luteinizing hormone or follicle-stimulating hormone analogs. Potential novel therapeutic agents include DNA, RNA, cytokines, peptide receptor antagonists, peptide receptor agonists, hormones, and enzymes. Nanotherapeutic treatment of testicular cancer, infertility, testicular torsion, orchalgia, hypogonadism, testicular infections, and cryptorchidism within the framework of potential target cells are an emerging area of research. While there are many potential applications of nanotechnology in drug delivery to the testis, this remains a relatively unexplored field. This review highlights the current status as well as potential future of nanotechnology in the development of novel therapeutics for testicular disorders.
KeywordsDrug delivery Testis Nanoparticles Infertility Sustained release
Before the first Pure Food and Drug Act in 1906, it was common for plasters, pills, and tonics to be erroneously marketed as having miraculous specificity for their target organs. For example, a mixture of sandal wood and saw palmetto, Sanmetto was marketed to have “special action upon the glands of the reproductive organs… its action is that of a great vitalizer, tending to increase their activity and to promote the secreting faculty” . Regrettably, a drug specifically targeted to the testis has not been so easy to achieve as mixing sandal wood and saw palmetto. In the past, drug delivery for testicular disorders has either been via oral or intravenous routes and has had systemic as well as local actions. Downsides of conventional drug delivery to treat testicular pathology include negative side effects of systemically administered drug, high dosing frequency, inability to achieve sufficiently high intratesticular concentrations for therapeutic activity, and failure of large molecules to cross the blood–testis barrier. Targeted therapies are currently limited or not advisable. For example, ultrasound-mediated transcutaneous drug delivery, i.e., sonophoresis, is limited to small molecules that would likely penetrate the skin in lesser amounts under passive conditions . Direct injection to the testis is contraindicated as the trauma to and disruption of the blood–testis barrier could induce fibrosis and anti-sperm antibody formation thereby decreasing fertility .
Summary of several testicular disorders, current treatments and potential applications of nanotechnology
Downsides of current therapy
Potential applications of nanotechnology
90–95% Germ cell tumors
Orchiectomy followed by surveillance, radiation, further surgery or chemotherapy
Imaging of micrometastatic disease
5–10% Stromal tumors
Localized delivery of toxic agents
Treatment of chemotherapy resistant tumors (i.e. teratoma) with toxic agents
Testis sparing treatment of testicular cancer (especially useful for CIS or patient with solitary testis)
Adoption, in vitro fertilization, or intrauterine insemination
Invasive, expensive, and increased risk of multiple gestations
GnRH, HcG, or recombinant FSH
Difficult dosing regimen
Stimulation of Leydig cells with LH analog or sustained release of testosterone in the testis
Disorders of spermatogenesis
Oral antioxidants, vitamins, antibiotics, or surgery
Secondary infections, antibiotic resistance, or testicular injury
Sustained localized delivery of antioxidants or antibiotics
Disorders of sperm delivery
Pseudoephedrine or surgery
Disorders of sperm function
Sustained localized delivery of anti-inflammatory agent without systemic side effects
Ischemia–reperfusion injury after testicle twists upon its cord
Surgical detorsion or testicle removal
Testicular atrophy or infertility
Sustained localized delivery of antioxidant or anti-inflammatory agent
Most prostate cancer requires androgen for growth
Hormones, hormone antagonists, anti-androgens, or surgery
Frequent dosing and adrenal insufficiency
Testis specific delivery of LH antagonist or inhibitors of steroid synthesis
Multiple etiologies including post surgical pain, post vasectomy pain, infection, varicocele, hydrocele, spermatocele, arteritis, or idiopathic
non-steroidal anti-inflammatories, tricyclic antidepressants, narcotics, anesthetic, or surgery
Poor efficacy Systemic toxicities:
Sustained release of nonsteroidal anti-inflammatory agents
Sustained localized delivery of anesthetics which would minimize potential cardiotoxicity
Sustained localized delivery of narcotics would eliminate systemic side effects as well as development of narcotic abuse/addiction
Infections: HIV, gonorrhea, epididymitis, orchitis
Antiretroviral therapy or antibiotics
Sustained localized delivery of antibiotic or antiretroviral across blood–testis barrier
Sustained localized delivery of analgesic along with active agent against infection
secondary infections (vaginitis, c diff colitis)
Testicular cancer is the most common cancer in 20 to 34-year-old men with an estimated 7,500 new cases of germ cell tumor diagnosed each year in the USA . Approximately 90–95% of testicular cancers are germ cell tumors, with the mainstays of therapy currently consisting of surgical removal of the testicle and spermatic cord through an incision in the groin followed by surveillance, surgical removal of metastatic lesions, radiation or chemotherapy depending on tumor characteristics and stage . While for most patients testicular removal is a viable option for diagnosis and treatment, this option is less than ideal in patients with either bilateral testicular tumors or a solitary testis. In these cases, some surgeons may perform testis-sparing surgery if the tumor is solitary, less than 2 cm in size, and confined to the testis . Cure rates for testicular cancer are excellent due to the exquisite sensitivity of testicular germ cell neoplasms to chemotherapy or radiotherapy. These rates depend on the stage and type of tumor with the lowest being a 25% overall cure rate for poor prognosis stage two to three nonseminomatous germ cell tumors versus >92% for good prognosis nonseminomatous germ cell tumors at the same stage and a >90% overall cure rate for seminomas . Unfortunately, some cancers do not respond to current therapies, and therapy becomes palliative. For the tumors that do respond, there are significant side effects to each of the therapeutic options including loss of fertility, myelosuppression, nausea, vomiting, hair loss, and late toxicities affecting the brain, ears, lungs, blood vessels, and kidneys. Secondary malignancies, one of the most concerning side effects, occur 1.43 times more often in testicular cancer patients having undergone systemic chemotherapy or radiation therapy and include leukemia, melanoma, non-Hodgkin's lymphoma, as well as cancers of the colon, stomach, kidney, prostate, bladder, thyroid, rectum, pancreas, and connective soft tissue . For men with metastatic chemotherapy-resistant tumors, novel toxic therapeutics could mean the difference between life and death. Conversely, for the men who can be cured with current treatment modalities, the development of novel drug delivery mechanisms could minimize systemic toxicities and secondary malignancies, making a significant contribution to the current armamentarium.
Male factor infertility can have a variety of etiologies. These can be divided into endocrine abnormalities, disorders of spermatogenesis, failure of sperm delivery, and abnormal spermatic function . For endocrine disorders including pituitary disease, Kallmann syndrome, follicle-stimulating hormone (FSH) deficiency, luteinizing hormone (LH) deficiency, androgen excess, congenital adrenal hyperplasia, estrogen excess, prolactin excess, corticosteroid use, thyroid abnormalities or endocrine receptor abnormalities, often treatment consists of correcting the underlying pathology or hormone supplementation. While attempting to correct hypogonadism often physicians will administer systemic testosterone, which regrettably actually causes or worsens infertility due to its feedback inhibition of testosterone secretion in the testis, where a high intratesticular level of testosterone is needed for induction and maintenance of spermatogenesis. Advantages of novel therapeutics would include targeting testosterone or LH analogs to the testis which would enable hormone repletion while avoiding the infertility seen with systemic administration. In addition, preparations of novel therapeutics with sustained release would eliminate difficulties with the frequent and or complicated dosing regiments that plague these men who often require life-long supplementation . While not possible with currently available delivery systems, gene therapy may be a potential treatment for those with receptor abnormalities . These endocrinopathies do not only affect men who are interested in fathering a child. Hypogonadism can be idiopathic or secondary to aging, narcotic use, renal insufficiency, diabetes mellitus, or cancer. While systemic therapy with testosterone is commonly used, this is less than ideal not only because of the infertility but also because of difficult dosing frequency and difficulty in obtaining adequate drug levels. Novel therapeutics similar to those suggested for infertility would be ideal in treating this patient population.
For some chromosomal disorders of spermatogenesis such as microdeletions found in the proximal or middle arm of the Y chromosome, there are no available treatment options for the patient's infertility. For those who are slightly more fortunate, including patients with Klinefelter's syndrome (karyotype 47XXY), microdeletions of the distal region of the long arm of the Y chromosome, Sertoli cell-only syndrome, or a history of chemotherapy or radiotherapy, surgical exploration of the testis can occasionally identify isolated areas of retained spermatogenesis . Other disorders of spermatogenesis can be seen due to a litany of causes including toxins such as heavy metals, occupational exposures such as heat, alcohol, cigarettes, or medication induced. While treatment for these disorders may include ART, an improvement in sperm quality and quantity may be seen after treatment with anti-estrogens which increase pituitary gonadotropic secretion, other gonadotropins, or a variety of vitamins, nutritional supplements, and anti-inflammatory agents . The therapeutic basis for vitamins, nutritional supplements, and anti-inflammatory agents lies in their antioxidant properties. Reactive oxygen species associated with a variety of the causes of male infertility, are thought to overwhelm the body's innate antioxidant mechanisms. Exogenous supplementation of the testes natural antioxidant defense mechanisms including; superoxide dismutase and catalase, may improve the fertility of this group of patients. For many patients, who are otherwise healthy and not taking medications, the dosing regimen of empiric vitamins, nutritional supplements, and anti-inflammatory agents can be difficult, leading to noncompliance. Targeting these agents to the testicular tissue with a sustained release formulation would theoretically lead to a decreased systemic dose and decreased dosing frequency, which would likely improve patient compliance. Sperm function disorders, including anti-sperm antibodies, can be treated with corticosteroids. Targeted local treatment, rather than systemic treatment with corticosteroids or other immune-modulating medications, would eliminate the toxicity of systemic immunosuppression. Finally, gene therapy may prove to be a potential avenue for treatment of patients with ultrastructural defects of the sperm.
Nanotechnology is an emerging field in the treatment of a plethora of medical problems. Using nanotechnology for drug delivery, highly water-insoluble or unstable products can be delivered to the patient. In theory, with tissue-specific targeting, the systemic toxicity of existing drugs can be reduced while the efficacy is improved. Multiple different nanotechnology-based drug delivery systems have been developed, including nanospheres, nanocapsules, ceramic nanoparticles, polymeric micelles, liposomes, dendrimers, and nanocrystals . While a discussion of the intricacies of these different drug delivery systems is outside the scope of this review, it is pertinent to note that for many of these systems, targeting and delivery is similar to that of nanoparticles. Nanoparticles are in the nanometer-sized range and have been used for drug delivery. For polymeric biodegradable nanoparticles, the drug of interest is dissolved, entrapped, absorbed, or attached into the nanoparticles. These agents are advantageous due to their small size, which can penetrate smaller capillaries, and are then taken up by cells via endocytosis. Release profiles can be modified by alterations in particle size, particle coating, and base polymer. For example, the ratio of poly dl-lactide co-glycolide and polylactide utilized in particle formation can be varied, resulting in a modifiable release profile due to differences in the hydrophobicity and degradation rate of the two polymers . Targeting to a particular cell type can be achieved by conjugation to a specific ligand, alterations of the characteristics of the nanoparticles including size, shape, charge and surface groups, or anatomically with injection into arteries feeding capillary beds of the target tissue . These various characteristics hold particular promise in overcoming the challenge of intracytoplasmic delivery of drug to specific cell types as well as the unique challenge of drug delivery across the blood–testis barrier.
There are two general approaches towards targeting therapy to different tissues: (1) active (i.e., ligand or physical external stimuli such as magnets) or (2) passive, which utilizes either anatomic or pathophysiologic factors . Anatomically directed delivery of nanoparticles (i.e., delivered to an artery that feeds a particular organ) minimizes the likelihood that the nanoparticles will be taken up by the reticulo-endothelial system (RES) prior to arrival at their target tissue. Injecting into the arterial supply of the target tissue avoids a first pass through the liver and spleen where systemically administered nanoparticles that have not had surface modification to prevent opsonization are removed from circulation by the RES within seconds . Another means of passive targeting takes advantage of the pathophysiology of the disease. For example, in inflammatory diseases and cancers, it has been demonstrated that vascular permeability is increased, thereby encouraging drug diffusion into tissues . Active targeting includes conjugating ligands, such as sugars, folic acid, peptides, or engineered antibodies . Given the different methods of targeting, it is necessary to understand the macroscopic anatomy (esp. vascular supply) as well as the microscopic and molecular scale of the target tissue in order to rationally design a novel drug delivery system.
Macroscopic testicular anatomy
The arterial blood supply to the testis is trifold and includes the testicular artery, which is a direct branch from the aorta; the vasal artery, which originates from the superior vesicle artery (a branch of the internal iliac); and the cremasteric artery, which arises from the inferior epigastric artery (a branch of the external iliac) . The testicular artery, the primary blood supply to the testis, branches into the internal, inferior, and capital arteries. This trifurcation occurs anywhere from the inguinal canal to the head of the epididymus . These arteries form a rich arterial supply to the testis, with the testicular and capital arteries anastamosing at the head of epididymis, and the epidymal, cremasteric and vasal arteries converging at the tail. Within the testicle, the testicular capillaries are located in the interstitium, and nutrients as well as hormones must diffuse through the extracellular matrix to the seminiferous tubules, the most metabolically active site in the testis . Testicular veins form the pampiniform plexus around the testicular artery allowing for a countercurrent heat exchange. This plexus coalesces into two to three veins at the level of the inguinal canal and finally into a single vein that either drains into the left renal vein or inferior vena cava for the left and right testicle, respectively.
Microscopic and ultrastructural testicular anatomy
FSH and LH are hormones secreted by the pituitary in response to gonadotropin-releasing hormone (GnRH) (Fig. 2). These hormones enter the circulation and bind to specific LH and FSH receptors in the testicular vascular endothelium which mediate rapid transcytosis (15 min) into the interstitium [24, 25]. Once in the testicular interstitium LH binds to the LH receptor, a G protein-coupled receptor, located on the Leydig cell surface causing increased testosterone synthesis. Interestingly, both the receptors for FSH and LH can be downregulated via internalization of the receptor and hormone by their respective cells . In Leydig cells, LH receptors are routed to lysosomes where both hormone and receptor are degraded, whereas in Sertoli cells, internalized FSH/FSH receptor complexes are internalized into endosomes and subsequently recycled whole back to the cell surface . Although previously thought to be only expressed by Sertoli cells, recent evidence has found that FSH receptors are also found on spermatogonia, where they also undergo similar receptor internalization and recycling .
Cell types and potential targets
The different testicular pathologies including infertility, oxidative stress from testicular torsion, testicular cancer, hypogonadism, exogenous steroid use, chronic orchalgia, testicular infections, or disorders of spermatogenesis caused by toxicant exposure, varicocele or aging can be categorized based on the types of cells involved. The pathologies, potential ligands, and what, if any, relevant nanotherapeutics currently exist will be categorized according to this schema.
Spermatogonia are the progenitor cells for germ cell tumors. These pluripotent cells can develop into a plethora of different morphologically distinct germ cell tumors including teratoma, seminoma, yolk sac tumor, embryonal carcinoma, choriocarcinoma, as well as mixed germ cell tumors . These germ cell tumors represent the overwhelming majority of testicular cancers (90–95%) with stromal tumors comprising the other 5–10% . In an effort to determine the potential toxicities of silver nanoparticles, one group documented the deleterious effects these nanoparticles have on spermatogonia . Unfortunately, anatomical targeting of these tissues via arterial infusion is a challenge as the testicular artery is small and difficult to access. Ligand active targeting may also be a challenge as, although normal spermatogonia express FSH receptors, at least one report suggests that testicular germ cell tumors may not . Interestingly, although potentially not demonstrated on germ cells themselves, FSH receptors have been shown to be present on endothelial cells of blood vessels closely associated with multiple different cancers including testicular, prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, and ovarian cancer . One group successfully utilized the fact that ovarian carcinoma cells express the FSH receptor to target paclitaxel-loaded nanoparticles to cancer in vivo with a FSH hormone peptide ligand. This group showed improved antiproliferation and antitumor effects with the FSH-conjugated nanoparticles as compared with either free paclitaxel or naked paclitaxel nanoparticles . Whether this effect was due to FSH receptors on the ovarian tumor cells themselves or the blood vessels surrounding the tumors is unclear. As mentioned earlier, the endothelium around and inside tumors is “leaky” which could also be utilized to target nanoparticles to tumors. Finally, while magnetic nanoparticles are not promising therapeutic agents for non-malignant diseases due to their inherent toxicity, they could be utilized with external magnets for specific targeting into testis tissue . These factors could be utilized to treat widely disseminated testicular cancer or carcinoma in situ; however, it is likely that radical orchiectomy will remain the treatment of choice for testicular tumors as it is both therapeutic and diagnostic.
As Sertoli cells are the chaperones of spermatogenesis and form the blood–testis barrier, they are rational targets for the development of reversible male contraception. The blood–testis barrier is a unique structure and therefore a potential site for creating specific ligands and drug targeting . Although the blood–testis barrier is located within the seminiferous tubule, ligand development to the blood–testis barrier may facilitate intracellular delivery of drug as endocytosis and recycling of junction proteins have been noted as part of the process allowing migration of spermatocytes to the adluminal compartment . Male contraceptive drugs could function by either causing premature release of germ cells which produces sperm unable to fertilize the ovum, or prolonged attachment which leads to phagocytosis of the degenerating germ cells by the Sertoli cells . To prevent potential formation of sperm auto-antibodies, altering the blood–testis barrier so that it cannot permit progression of spermatocytes would be the preferable option. Theoretically, a potential male contraceptive medication that affects the blood–testis barrier, such as chloroquine, could be targeted to the Sertoli cells either via FSH hormone or ligands specific to the blood–testis barrier . A previously developed male contraceptive, Adjudin, perturbs Sertoli germ cell adhesion reversibly. This is not currently used because the margin between safety and efficacy is too narrow; however, with specific delivery to the testis and sustained release, this margin may easily be widened . As our understanding of the ultrastructural composition and regulation of the blood–testis barrier is further elucidated, cytokines that play an important role in its function could be packaged into nanoparticles and delivered to Sertoli cells as novel contraceptives.
Given that Sertoli cells secrete many of the nutrients and factors necessary for the survival and differentiation of germ cells; they are logical locations for delivery of antioxidant medications. Testicular oxidative stress is a common feature of several pathologic conditions including male infertility, testicular torsion, diabetes mellitus, toxicant exposure, varicocele, and aging [40, 41]. One group administered hydrated C60 fullerenes, a powerful bioantioxidant, without any targeting moiety and was able to protect reproductive function in rats with diabetes-induced testicular oxidative stress . Testicular torsion, another cause of testicular oxidative stress, occurs in as many as one in 158 males by the age of 25 and is commonly associated with testicular atrophy and infertility despite prompt surgical detorsion [43, 44]. Torsion is an ischemic–reperfusion injury, with damage induced by excessive production of reactive oxygen species during the reperfusion phase. A primary human defense against damage from reactive oxygen species is the antioxidant enzyme superoxide dismutase. Unfortunately, the half-life of superoxide dismutase is only 6 min [45, 46]. By encapsulation into nanoparticles, the effective half-life of superoxide dismutase could be significantly extended. This property could be synergistically enhanced by selective targeting, which could theoretically widen the window for testicular salvage. Oral antioxidants have been shown to quadruple the spontaneous pregnancy rate and reduce the cost per pregnancy by 60% in one report . Perhaps with selective targeting to Sertoli cells with either superoxide dismutase or other antioxidant nanotherapeutics, the impact of testicular oxidative stress could be significantly reduced.
Leydig cells respond to LH and produce testosterone; therefore, they are potential targets for inducing or correcting hormonal disorders. A common clinical situation where a physician may wish to induce a hormone disorder is with androgen ablation in the treatment of metastatic prostate cancer. In men with metastatic prostate cancer, androgen blockade reduces cancer growth rate and has been accomplished by multiple methods including medical treatment with estrogens, progestins, prolactin antagonists, LHRH agonists, GnRH antagonists, inhibitors of androgen synthesis, anti-androgens, as well as surgically with bilateral orchiectomy. Nanoparticles targeted to Leydig cells, via a LH analog, could potentially block testosterone synthesis either reversibly or irreversibly, which could represent another potential treatment for metastatic prostate cancer.
Male hypogonadism, i.e., testosterone deficiency, can be the result of multiple different disease states including primary hypogonadism (Klinefelter's syndrome 47, XXY), secondary hypogonadism (Kallman's syndrome), chemotherapy or radiotherapy exposure, as well as an unfortunate result of aging . Symptoms of hypogonadism include infertility, decreased libido, fatigue, cognitive deficiency, erectile dysfunction, osteopenia, decreased muscle mass, and change in fat distribution. This is a widespread problem affecting approximately 70% of men aged 70–79 . For many of the causes of hypogonadism, stimulating the Leydig cells to secrete more testosterone could be an effective treatment.
Another cause of infertility is exogenous steroid use. Elevated systemic testosterone levels induce negative feedback onto the pituitary and Leydig cells, thereby decreasing the intratesticular testosterone levels which are critical for the induction and maintenance of spermatogenesis. Recovery of spermatogenesis, if it occurs, is often prolonged. Stimulating Leydig cells themselves could help treat illicit exogenous steroid abuse as well as potentially eliminate the infertility that is seen as the unintended effect of testosterone repletion by physicians. In order to increase Leydig cell production of testosterone, an active LH analog would make a rational therapeutic as well as targeting ligand. In patients in whom the Leydig cells are incapable of responding, nanotherapeutic agents targeted to the testis may be able to deliver high levels of local testosterone therein facilitating recovery of spermatogenesis.
Capitalizing on the fact that endothelial cells of the testicular micro-capillaries express both the FSH and LH receptors, one could target anti-inflammatory, neuromodulatory, or antibiotic drugs for the treatment of pain syndromes including chronic orchalgia or testicular infections while minimizing systemic effects of the drugs. Treatment of HIV and prevention of the transmission of HIV via sexual contact may be improved by seminiferous tubule delivery of anti-HIV drugs. With the increase in utilization of highly active antiretroviral therapy (HAART), there has been an increase in the number of patients who develop resistance strains of HIV . One likely cause of increased resistance is due to the low concentrations of HAART drugs in the semen and seminiferous tubules, a privileged site due to the blood–testis barrier. It is known that delivery of these drugs to the seminiferous tubules requires passage through the blood–testis barrier which is a barrier that many HAART drugs including enfuvirtide and many protease inhibitors are unable to pass . Additionally, disorders that historically were repaired with surgery, such as undescended testis, may be able to be treated medically with nanotherapeutics. For example, encapsulation of factors such as insulin-like peptide three and stimulation of increased testosterone secretion by Leydig cells may encourage testicular descent in cryptorchid testis .
Further research in drug delivery for testicular disorders is made possible by a grant from the Cleveland Clinic Foundation Research Program Committee.