Letter pubs.acs.org/NanoLett
Achieving a New Controllable Male Contraception by the Photothermal Effect of Gold Nanorods Wen-qing Li,† Chun-yang Sun,† Feng Wang,‡ Yu-cai Wang,‡ Yi-wen Zhai,† Meng Liang,† Wen-jing Liu,† Zhi-min Liu,† Jun Wang,*,†,‡ and Fei Sun*,†,‡ †
Department of Cell and Developmental Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China ‡ Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, Anhui 230027, China S Supporting Information *
ABSTRACT: During the process of human civilization, owning household pets has become increasingly popular. However, dogs and cats may be reservoirs or vectors of transmissible diseases to humans. Confronted with the overpopulation of pets, traditional contraception methods, surgical methods of sterilization, for animals are used, namely, ovariohysterectomy and orchidectomy. Therefore, a simple, nonsurgical, controllable, more effective and less expensive contraception method is highly desirable. In this study, we show that in situ testicular injection of methoxy poly(ethylene glycol)-modified gold nanorods with near-infrared irradiation in male mice can achieve short-lived or permanent male infertility. In a lower hyperthermia treatment, the morphology of testes and seminiferous tubules is only partly injured, and fertility indices are decreased to ∼10% at day 7, then recovered to 50% at day 60. In a higher hyperthermia treatment, the morphology of testes and seminiferous tubules are totally destroyed, and fertility indices are decreased to 0 at day 7. Overall, our results indicate a potential application of plasmonic nanomaterials for male contraception. KEYWORDS: Gold nanorods, photothermal effect, male contraception, fertility
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at day 7, then recovered to 50% at day 60. In a higher hyperthermia treatment, the morphology of testes and seminiferous tubules are totally destroyed, and fertility indices are decreased to 0 at day 7. Overall, our results indicate a potential application of plasmonic nanomaterials on male contraception. GNR, known to be relatively stable and biocompatible,8 were obtained by the modification of gold nanorods with α-lipoyl-ωhydroxyl poly(ethylene glycol) (Mw = 5000) through ligand exchange from acetyl trimethylammonium bromide (CTAB)stabilized gold nanorods.9,10 As shown in Figure 1a, GNR was shown by TEM to have rod morphology with dimensions of ∼40 nm × 10 nm (aspect ratio of ∼4). Fourier transform infrared (FITR) analysis indicated the existence of a methoxy poly(ethylene glycol) (PEG) shell due to the appearance of a CO stretching vibration band at 1720 cm−1, specifically assigned to PEG. The UV−vis spectra demonstrated that GNR had an apparent surface plasmonic resonance band at ∼800 nm in the near-infrared region (Figure 1b), favoring its photothermal transfer in deep tissue.11,12 To investigate the temperature response of an aqueous suspension of GNR to
uring the evolution of human civilization, humans have adopted more and more pets. Over half of all households in the United States own a dog or cat.1 Although the exact figures are unknown, the Humane Society of the United States estimates that between 8 and 10 million dogs and cats enter shelters each year and 4−5 million of these animals are euthanized.2 Dogs and cats, many of them unwanted, may be reservoirs or vectors of transmissible diseases to humans. Confronted with the overpopulation of pets, using traditional contraception methods for animals, surgical methods of sterilization, namely, ovariohysterectomy (spaying) and orchidectomy (castration), is necessary.3 Therefore, a simple, nonsurgical, controllable, more effective and less expensive contraception method is highly desirable. Male fertility is temperature-dependent, as hyperthermia will destroy testicular function and even injure spermatogenesis and male infertility.4 As a photothermal transducer, gold nanorods have been used for the photothermal therapy of cancers.5−7 We hypothesize that the controllable male infertility may be achieved by controlling the photothermal effect of gold nanorods. Here, we show that in situ testicular injection of methoxy poly(ethylene glycol)-modified gold nanorods (here abbreviated GNR) with near-infrared (NIR) irradiation in male mice can achieve shortlived or permanent male infertility. In a lower hyperthermia treatment, the morphology of testes and seminiferous tubules is only partly injured, and fertility indices are decreased to ∼10% © XXXX American Chemical Society
Received: February 11, 2013 Revised: May 17, 2013
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Figure 1. Preparation and characterization of methoxy poly(ethylene glycol)-modified gold nanorods (GNR). (a) Scheme of structure and TEM image of GNR (scale = 50 nm). (b) Extinction spectrum of GNR in water. (c) Temperature change curves of GNR aqueous solutions treated with different NIR irradiation and various GNR concentrations.
testes continued to increase in the first 4−5 min and was not significantly changed after that period, reaching an average temperature of 31, 37, and 40 °C for PBS+NIR, 105 μM GNR +NIR, and 145 μM GNR+NIR treated with 0.8 W/cm2, respectively. When the NIR irradiation density was elevated to 1.0 W/cm2, the average temperature of testes reached 45 °C for 145 μM GNR treated with 1.0 W/cm2. The above results suggested the controllable temperature of testes by adjusting the concentration of GNR and NIR irradiation density in vivo, indicating that controllable male contraception may be obtained. During in vivo experiments, the male ICR mice were randomly divided into six groups which were treated as mentioned above. In order to highlight the temperature of GNR+NIR treatment, we recorded PBS (29 °C), 145 μM GNR (28 °C), PBS+NIR (31 °C), 105 μM GNR +NIR (37 °C), 145 μM GNR+NIR (40 °C), and 145 μM GNR +NIR (45 °C). Experimental mice were housed for 7, 30, and 60 days and sacrificed for further investigation. Throughout the entire experimental period, none of the mice in any group showed stress. We supervised these mice daily and did not observe any abnormal behaviors among the groups. The body weights were not significantly different among 145 μM GNR (28 °C), PBS+NIR (31 °C), 105 μM GNR+NIR (37 °C), 145 μM GNR+NIR (40 °C), and 145 μM GNR+NIR (45 °C) when compared to PBS (29 °C) at each time point (Figure 2c). The testes were destroyed partially by treating with 105 μM
the irradiation of a diode laser at 808 nm, we measured the temperature changes of the suspension at different time points. As depicted in Figure 1c, phosphate-buffered saline (PBS) did not show a response to the irradiation even at 1.0 W/cm2, while the temperature of the aqueous suspension of 145 μM GNR (by Au atom) increased with the extension of irradiation time, reaching 46 and 52 °C in 10 min at the irradiation density of 0.8 W/cm2 and 1.0 W/cm2, respectively. Irradiation to 105 μM GNR led to lower temperatures at 10 min, reaching 43 and 48 °C at 0.8 W/cm2 and 1.0 W/cm2, respectively, suggesting that the temperature can be easily controlled by adjusting the concentration of GNR and NIR irradiation density. We next examined the photothermal effect of GNR dose and the NIR irradiation density via in vivo studies. The male ICR mice were randomly divided into six groups, which were PBS, 145 μM GNR, PBS+NIR 0.8 W/cm2, 105 μM GNR+NIR 0.8 W/cm2, 145 μM GNR+NIR 0.8 W/cm2, and 145 μM GNR +NIR 1.0 W/cm2. The mice received a single testicular injection with PBS, 105 μM GNR, or 145 μM GNR. Then, the testes of mice were exposed for up to 10 min with the diode laser at 808 nm at a power density of 0.8 W/cm2 or 1.0 W/cm2 (Figure 2a). The temperature of a circular region of interest (ROI) encompassing the irradiated testes, recorded by an infrared camera, is shown in Figure 2b. The average temperatures of testes would only remain at 29 and 28 °C in PBS and 145 μM GNR-treated mice only. The temperature of B
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Figure 2. In vivo photothermal treatment of mice with GNR+NIR. (a) Thermal infrared images of mice testes with GNR injection recorded at different time intervals. The mouse testis was exposed to NIR laser at a power density of 0, 0.8, or 1.0 W/cm2. (b) Plots of temperature of mouse testes with GNR injection as a function of irradiation time. The mouse testis was exposed to NIR laser at a power density of 0, 0.8, or 1.0 W/cm2. (c) Body weights of treated mice. Data are shown as mean ± SEM (n = 5).
GNR+NIR (37 °C), and 145 μM GNR+NIR (40 °C), in comparison with PBS at 7 d (Figure 3a). Figure 3a also shows
that the testes were totally destroyed by treating with 145 μM GNR+NIR (45 °C) compared to PBS (29 °C) at 7 days, and C
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Figure 3. Impact on spermatogenesis after GNR+NIR treatment with 0.8 W/cm2 or 1.0 W/cm2 irradiation for 10 min. (a) Images of testes of GNR +NIR treated mice recorded at the 7th day following NIR laser irradiation (scale = 5 mm). (b) Testis indices and epididymis indices of GNR+NIR treated mice (n = 5). Testis indices and epididymis indices represent the weight percentage of testis and epididymis to the mouse body weight, respectively. Data are shown as mean ± SEM. NU indicates there was none after treatment with 145 μM GNR+NIR 1.0 W/cm2 (45 °C). (c) H&E stained testicular cross sections of GNR+NIR treated mice at 7, 30, and 60 days (scale = 100 μm).
μM GNR+NIR (40 °C), and 145 μM GNR+NIR (45 °C) in comparison to PBS (29 °C) at each time point. However, the testes or testis indices were not significantly different among the 145 μM GNR (28 °C), PBS+NIR (31 °C), and PBS (29
the testes disappeared by treating with 145 μM GNR+NIR (45 °C) at 60 day. Meanwhile, the results showed testis indices (weight percentage of testis to the mouse body weight) were decreased by treating with 105 μM GNR+NIR (37 °C), 145 D
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Figure 4. Fertility of the mice after GNR+NIR treatment with 0.8 W/cm2 or 1.0 W/cm2 irradiation for 10 min. Fertility index of male mice and the average number of pups/pregnant females after 7 and 60 days treatment with GNR+NIR (n = 5 or 6 in each group). NU indicates there was none after treatment with 145 μM GNR+NIR 1.0 W/cm2 (45 °C).
showed no significant differences among 145 μM GNR (28 °C) and PBS+NIR (31 °C) when compared with PBS (29 °C) at each time point (Figure 3c). In addition, histological examination of epididymis showed that when compared with PBS (29 °C) treatment sperm disappeared at 7 days after treatment with 105 μM GNR+NIR (37 °C) or 145 μM GNR +NIR (40 °C), but the sperm was recovered at 30 and 60 days. However, treatment with 145 μM GNR+NIR (45 °C) led to a complete disappearance at all of the observed time points (Supporting Information, Figure S1). The regulation of spermatogenesis of the pituitary gland was first described by Smith in 1927. His findings provide the basis for the current understanding of dual control of the endocrine and spermatogenic functions of the testes by pituitary luteinizing hormone (LH) (via production of testosterone) and follicle-stimulating hormone (FSH).19 For example, LH acts on the Leydig cells of the testes to stimulate them to synthesize and secrete the male sex hormone testosterone (T), and pituitary FSH stimulates the Sertoli cells to produce androgen-binding protein and the formation of the connections between Sertoli cells that make up the BTB.20 Alteration of the balance of hormone levels will result in testicular damage, malfunction of spermatogenesis, and male infertility. In the present study, plasma levels of testosterone (T), LH, and FSH were determined by enzyme-linked immunosorbent assay (ELISA). The results indicated that levels of plasma FSH, LH, and T had no significant differences among all groups at each time point (Supporting Information, Figure S2). Previous studies showed no significant changes of blood testosterone levels due to warm water, infrared, microwaves, or ultrasound heat treatment after 2 weeks and after 60 days; this observation was also confirmed after 10 months of heat treatment.13
°C) groups (Figure 3b). The epididymis indices (weight percentage of epididymis to the mouse body weight) did not differ among PBS (29 °C), 145 μM GNR (28 °C), PBS+NIR (31 °C), 105 μM GNR+NIR (37 °C), and 145 μM GNR+NIR (40 °C) groups, but the epididymis disappeared in 145 μM GNR+NIR (45 °C) group at 60 day (Figure 3b). In addition, there were no changes in male mouse behaviors, including eating, drinking, sex drive, and fighting. Meanwhile, the data suggested that GNR might be safe with regard to testes function. Although some studies reported that only higher power IR irradiation would destroy the testes of mice,13 our results showed that, under a lower power, NIR irradiation based on photothermally converted nanomaterials would be safer and more effective. Previous studies reported that the hyperthermia of testes caused the degeneration of spermatogenic (mainly spermatocytes and spermatozoa) cells in seminiferous tubules.4,13−15 Figure 3a shows that the testes of GNR+NIR treated-mice were destroyed partially or totally, which indicated that spermatogenesis may be destroyed. Here, histological inspections of testes samples were performed. Figure 3c shows that the disordered and degenerated spermatogenic cells were mainly spermatocytes and spermatozoa caused by treatment with 105 μM GNR+NIR (37 °C) and 145 μM GNR+NIR (40 °C), when compared with PBS (29 °C) at each time point. After 145 μM GNR+NIR (45 °C) treatment, spermatogenic cells were disordered and completely degenerated (Figure 3c). The results were as the same as previous reports, which showed pachytene spermatocytes, early spermatids, and sperm are the cell types that are most susceptible to testicular heat stress.16−18 The spermatogonia and spermatogonial stem cells may not be destroyed by lower hyperthermia of GNR+NIR treatment. Moreover, the spermatogenic cells in seminiferous tubules E
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Figure 5. Coomassie brilliant blue staining and Western blotting analyses of apoptosis-associated proteins in mice after GNR+NIR treatment with 0.8 W/cm2 or 1.0 W/cm2 irradiation for 10 min. (a) Total proteins of testes analyses through Coomassie brilliant blue staining after 7 days of GNR +NIR treatment. (b) Western blotting analyses of apoptosis-associated proteins after 7, 30, and 60 days of GNR+NIR treatment.
cell types that are most susceptible to testicular heat stress.16−18 Therefore, it is reasonable to state that the testicular disorder during a lower hyperthermia GNR+NIR treatment might be recovered after a long period because the superior spermatogonia and spermatogonial stem cells which possess the differentiation capacity were preserved. In addition, among all of the groups, pregnant female mice gave birth to almost the same numbers of pups (Figure 4), and all of the pups had no visible morphological defects. These data suggested that GNR themselves have no toxicity to the male reproductive system. This biocompatibility of GNR offered prerequisites for application in the male reproductive system. The effective contraception of male mice supplied prospects for application in the male reproductive system. Next, we explored the pathway of the photothermal effect of GNR+NIR on spermatogenesis. Wang et al. reported that hyperthermia of testes would induce apoptosis of sperm cells.21,22 However, the mechanism of photothermal therapy of GNR to cancers was involved in protein denaturation-triggered
Therefore, our results were consistent with the previous study and were better than castration with regard to sex levels. Fertility assessments were carried out on day 7 and 60 after GNR+NIR treatment. Each male mouse treated with GNR was paired with two virgin untreated female ICR mice in an individual cage. The fertility indices were decreased to ∼10% by treatment with 105 μM GNR+NIR (37 °C) and 145 μM GNR +NIR (40 °C) compared to PBS (29 °C) after 7 days (Figure 4), but they were recovered to 50% after 60 days (Figure 4). In a higher hyperthermia treatment of 145 μM GNR+NIR (45 °C), fertility indices were decreased to 0 by treating with 145 μM GNR+NIR (45 °C) compared to PBS (29 °C) after 7 days (Figure 4). The data showed that the fertility index was dependent on hyperthermia GNR+NIR treatment. The results indicated that the fertility of mice can be well-controlled by hyperthermia treatment of GNR+NIR. Noticeably, the fertility was normal among 145 μM GNR (28 °C), PBS+NIR (31 °C), and PBS (29 °C) treated mice. It has been confirmed that pachytene spermatocytes, early spermatids, and sperm are the F
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cellular injury/death.23,24 In this study, all proteins of the testes in each group were examined by SDS-PAGE. The results showed that testicular proteins were not significantly different among all groups treated with 145 μM GNR (28 °C), PBS +NIR (31 °C), 105 μM GNR+NIR (37 °C), and 145 μM GNR +NIR (40 °C) at day 7 (Figure 5a), but testicular proteins were degenerated by treating with 145 μM GNR+NIR (45 °C) compared to PBS (29 °C) at day 7 (Figure 5a). We next needed to prove whether the destruction was a result of apoptosis by analysis of PARP and Caspase 3 (apoptosis markers). Figure 5b shows the total PARP proteins disappeared in the testes treated with 145 μM GNR+NIR (45 °C) and decreased in the testes treated with 145 μM GNR+NIR (40 °C) in comparison with PBS (29 °C) at day 7, while the active PARP showed no significant differences among all groups. Interestingly, after treatment with 145 μM GNR+NIR (40 °C), the total PARP was recovered to normal levels at day 60 (Figure 5b). The Caspase 3 showed no significant differences among all groups (Figure 5b). A previous study showed a decrease of total PARP proteins, which indicated protein degradation.25 In this study, the results suggested that testicular injury caused by hyperthermia of GNR+NIR was mainly involved in protein degradation. We have also investigated the gold accumulation in different tissues at day 7, 30, and 60. Following the GNR+NIR treatment, mice were sacrificed, and heart, liver, spleen, lung, kidney, testis, epididymis, and blood were extracted and analyzed by ICP-MS to measure the accumulated gold. It showed that the gold was mainly distributed in liver, spleen, testis, and epididymis (Supporting Information, Figure S3). The gold concentration in the testis and epididymis continued decreasing with the extended time, but slightly increased in the spleen. In a previous report, PEG-modified gold nanorods (PEG-NRs) were found to clear via reticuloendothelial system (RES) uptake with splenic clearance following systemic injection, and results indicated that the organs of the reticuloendothelial system (the liver and spleen) had substantial nanorod accumulations.9,26 In this study, macrophage-like cells in the testes may capture GNR and transport it into blood immediately after testicular injection, which was further cleared by RES. We have demonstrated a new male contraception via the photothermal effects of gold nanorods. The contraception strategy was based on the mechanism of testicular protein denaturation, including the in situ testicular injection of GNR with appropriate near-infrared (NIR) irradiation to produce photothermal effects in male mice. In the higher hyperthermia states of GNR+NIR, permanent infertility of males was obtained, similar to “castration”. In the lower hyperthermia states of GNR+NIR, short-lived infertility was achieved. Herein, we have reported a less invasive and controllable procedure for male contraception. This method is more convenient, efficient, and cheaper than surgical castration, which can be chosen to treat the overpopulation of pets and stray animals. Overall, our results indicated the potential application of plasmonic nanomaterials to male contraception. Considering the highly diverse concentration of plasmonic nanomaterials, power density and the duration of laser irradiation, and the multiple ways in which animal exposure to plasmonic nanomaterials can occur, further studies on the optimization of the conditions of plasmonic nanomaterials are urgently needed.
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ASSOCIATED CONTENT
S Supporting Information *
Materials, detailed experimental methods, supplementary figures, and supplementary references. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (J.W.);
[email protected] (F.S.). Author Contributions
W-Q. Li and C-Y. Sun contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the following grants: the National Basic Research Program of China (2009CB941700, 2010CB934001); the National Natural Science Foundation of China (81125005, 30971091 and 51125012); the Chinese Academy of Sciences Knowledge Creative Program (KSCX2EW-R-07); the Fundamental Research Funds for the Central Universities (WK2070000008).
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ABBREVIATIONS PEG, poly(ethylene glycol); GNR, poly(ethylene glycol)modified gold nanorods; NIR, near-infrared; PBS, phosphatebuffered saline
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