Review Review

For reprint orders, please contact: reprints@futuremedicine.com

Nanoparticles and spermatogenesis: how do nanoparticles affect spermatogenesis and penetrate the bloodtestis barrier
Due to the widespread use of nanomaterials in medical, industrial and military applications, the question as to whether nanoparticles (NPs) cause harmful disturbances in human health, especially on the reproductive system, remains a matter of concern. In this review, we focus mainly on the invivo and invitro effects of NPs on spermatogenesis at the clinical, cellular and molecular levels. In general, most NPs display adverse effects on spermatogenesis at these various levels; but, some NPs show no adverse effects. However, the mechanism underlying NP disruption of spermatogenesis and penetration of the bloodtestis barrier remains unclear. In this review, we raise many hypotheses for experimental testing in order to elucidate the mechanism.
KEYWORDS: bloodtestis barrier n human health n nanoparticles n spermatogenesis

Zhou Lan & Wan-Xi Yang*

Nanotechnology is a rapidly emerging industrial area that studies and develops materials with surface structure and chemical properties on the nanoscale dimension and with special properties arising from this. One of the definitions of the term nanoscale is particles that are <100nm in at least one dimension [1] and which are therefore called nanoparticles (NPs). However, some particles that are >100nm but <1000nm are also called NPs if the particles demonstrate some special properties. For example, recent advances in nanotechnology allowed the production of a unique copper nanostructure that combines two special properties of strength and ductility that are often mutually exclusive [2] . In the past decade, the nanotechnology industry has grown rapidly worldwide and predictions indicate continued growth in the future. The cause of this rapid growth involves the ever-expanding applications of nanomaterials/NPs in the following areas: catalysts, fuel lubricants, paints, conductive inks, cosmetics, drug delivery, hydrogen storage, sensor devices, structural materials, medical therapeutics, UV light absorption, and free-radical scavengers [3] . Recently, the applications of NPs have expanded to include measurement science [4] , dermatology [5] , cancer therapy [6] and even cosmetics [7] . This rapid growth calls into question not only the health of workers at the manufacturing plant but also the health of the public due to the release of nanomaterials/NPs in the environment. Stern and McNeil reviewed NP safety issues and identified areas of agreement and disagreement with regard to NP risk, NP exposure and
10.2217/NNM.12.20 2012 Future Medicine Ltd

NP hazards [8] . They not only indicated that a paucity of NP safety studies exist but also that the NP safety studies are predominantly acute toxicology studies, not chronic studies. In addition, the current paradigm of NP safety focuses only on NP size as the determining factor in toxicity to the exclusion of other important NP properties (e.g., surface structure, surface chemistry, inorganic or organic surface coatings) that significantly change NP biocompatibility. Other studies and reviews have also addressed the issue of safety concerns and NP toxicology[914] . Epidemiologists have taken an active interest in male reproductive health because young men in some regions demonstrate suboptimal sperm quality and sperm number [15,16] . The alarming fact is that both human sperm quality and sperm number have declined in recent years and the cause is unknown in the majority of cases. The possibility of public exposure must be considered. Hoover etal. indicates that the potential routes of exposure to NPs include inhalation, dermal and possibly ingestion exposures, and that a wide range of NP sizes, shapes, functionalities, concentrations, exposure frequencies and durations are involved [17] . Despite the abovementioned findings, the role that NPs play as a reproductive toxicant in spermatogenesis remains largely unknown. In this regard, Ema etal. reviewed the reproductive and developmental toxicity of NPs [9] . Ema etal. indicated that invivo studies show that titanium dioxide (TiO2) NPs injected subcutaneously into pregnant mice result in male offspring with altered spermatogenesis and histopathological changes in the testes, and that carbon black (CB) NPs instilled within
Nanomedicine (2012) 7(4), 579596

The Sperm Laboratory, College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, Zhejiang, 310058, PR China *Author for correspondence: Tel.: +86 571 8820 6643 Fax: +86 571 8820 6485 wxyang@spermlab.org

part of

ISSN 1743-5889

579

Review

Lan & Yang

the trachea of pregnant mice result in male offspring with altered spermatogenesis [9] . In addition, Ema etal. indicated that invitro studies show that: TiO2 and CB NPs affect Leydig cell viability; gold NPs reduce human sperm motility; and silver, aluminum and molybdenum trioxide NPs damage spermatogonia stem cells [9] . However, we admit that no obvious clinical evidence shows a direct causeeffect relationship between the problem of human sperm quality/ quantity and NPs. Cormier etal. pointed out that particles will affect the human reproductive system, but what kind of particles (NPs or nonNPs) affect the human reproductive system were not pointed out [18] . The fact that the problem of human sperm quality and quantity could be caused by multiple factors makes it impossible to come to an absolute conclusion. However, the many experimental results on model animals and considerable exposure of humans to NPs make it reasonable for us to consider that NPs may be one of the factors. Bonde etal. [19] points out that less-appropriate parameters, incomplete data and insufficient statistical methods have led to the inaccurate conclusion that the quantity as well as the quality of sperm has been declining, which was made by Farrow in 1994 [16] . Bonde etal. also thought that not only environmental toxicants, but also the wide range of behavioral, medical and other factors that have the potential to damage human reproduction should be taken into consideration [19] . Our conclusion is, that according to the previous result that NPs affect animal spermatogenesis and the fact that NPs exist in our environment, we can at least give a warning that NPs may be a threat to people who are largely exposed to them. By contrast, not all NPs will necessarily demonstrate an adverse effect leading to toxicity. For example, Shi etal. reported that nanoselenium (nano-Se) diet supplementation produced positive effects on various parameters of sperm quality in male goats [20] . Thus, NPs must be investigated on a case-by-case basis without a predetermined bias as to whether a NP will have a positive or negative effect on a particular system or parameter. In addition, spermatogenesis occurs in a very safe environment because of the bloodtestis barrier (BTB) [21] . NPs, theoretically speaking, cannot penetrate the BTB. However, the following studies discussed in this article show that NPs have the capacity to penetrate the BTB while non-NPs do not. In order to summarize the extent of our knowledge at the present time, this review
580
Nanomedicine (2012) 7 (4)

mainly focuses on how NPs affect spermatogenesis at the clinical, cellular and molecular levels, and how NPs penetrate the BTB.

How do NPs affect spermatogenesis? Many recent reports indicate that most NPs show an adverse or toxic effect on spermatogenesis. By contrast, some NPs show a beneficial or nontoxic effect on spermatogenesis [20,22] . In order to study the invivo effects of NPs on spermatogenesis, administration via oral, air and serum routes were used. In order to study the invitro effects of NPs on spermatogenesis, various cultured cell lines were used. In general, these reports indicate that the species of animal, route of administration, dose of NP and NP characteristics (e.g., size, shape, chemical composition, surface area and surface charge) play a significant role in determining the effect of NPs on spermatogenesis [23] .
Invivo studies
Clinical level

The study of NPs and spermatogenesis at the clinical level measures body weight, organ weight, oxidative stress indicators and NP concentration/content indices, generally in mice or rats. The detailed methodology in such studies follows the generally accepted and newly revised toxicological research protocols [24] .
Nano-Se

The Swedish chemist Jons Jacob Berzelius first isolated and identified the trace element Se in 1817 [25] . Since then, reports concerning Se have indicated contradictory properties ranging from a nontoxic, essential element for mammalian health [26] to a toxic element [27] . Nano-Se, in the presence of a biological macromolecule such as protein, usually acts as a dispersion agent [28] . In this regard, animal studies show an increased toxicity of inorganic Se compared with selenate and elemental Se, including nano-Se. In addition, selenocysteine (an organic form of Se) and selenite demonstrate an increased toxicity compared with selenomethionine (also an organic form of Se). Jia etal. studied rats fed diets containing nano-Se, selenite, and high-Se protein at various Se concentrations: 0 (control group), 2, 3, 4 and 5ppm Se for 13weeks [29] . They reported significant toxicity as measured by body weight, hematology, clinical chemistry, relative organ weights and histopathology parameters in all three diets only at the higher doses of 4 and 5ppm Se. However, the toxicity in the rats fed nano-Se at
future science group

How nanoparticles affect spermatogenesis

Review

4 and 5ppm Se was less pronounced compared with rats fed selenite or high-Se protein diets at 4 and 5ppm Se. Shi etal. studied Taihang goats fed diets containing nano-Se, selenite and Se-yeast at Se concentrations of 0 (control group) and 0.3mg/kg Se for 90days [20] . They found that the final body weight increased in all goats fed Se compared with the control goats; average daily weight gain was greater in the goats fed nanoSe and Se-yeast diets compared with goats fed selenite and control diets; and Se content of the testes in the goats fed nano-Se was higher compared with goats fed selenite or Se-yeast diets. Later, Shi etal. obtained similar findings using Boer goats [22] . The antioxidant enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase eliminate reactive oxygen species. In addition, malondialdehyde (MDA) is an oxidative end product that serves as an index of antioxidant status. In the study on Taihang goats, Shi etal. reported that the serum activity of GSH-Px, SOD and catalase was higher in goats fed nano-Se compared with goats fed selenite or Se-yeast diets [20] . In addition, they found that the MDA levels in the goats fed nano-Se were the lowest compared with goats fed selenite or Se yeast diets or controls. This indicates that nano-Se has strong antioxidant properties. Later, Shi etal. obtained similar findings using Boer goats [22] . The cellular level of nano-Se is discussed in Nano-Se below.
Nano-Au

The use of medicinal gold dates back to the Vedic age, when colloidal gold (called Swarma Bhasma or gold ash) was employed in Ayurvedic medicine for its rejuvenation effects. At present, nano-Au is used as a drug carrier [30] , a photothermal agent for cancer therapy [31] and a treatment for rheumatoid arthritis [32,33] . In general, a myriad of studies indicate that the nano-Au biodistributes in both a time-dependent and a size-dependent manner. In order to address the time-dependent manner of nano-Au biodistribution, Balasubramanian etal. studied rats that were injected intravenously with a single 0.01-mg/kg body weight dose of 20-nm AuNPs and sacrificed at 1day, 1week, 1and 2months postinjection [34] . They found no significant accumulation of AuNPs in the testes at 1day or 1week postinjection versus the control. However, a significant accumulation of AuNPs occurred
future science group

in the testes at 1 (0.60.2ng/g tissue) and 2months (0.60.1ng/g tissue) postinjection versus the control (0.40.2ng/g tissue). This suggests that AuNPs biodistribute to the testes in a time-dependent manner. In this same study, AuNPs accumulated to the highest extent in the liver and spleen, probably due to the sinusoidal nature of the capillary network and the presence of a significant macrophage population in bothorgans. In order to address the size-dependent manner of nano-Au biodistribution, De Jong etal. studied rats that were injected intravenously with a single dose of 10-, 50-, 100- or 250-nm Au particles and sacrificed at 1day postinjection [35] . They reported that a significant accumulation of 10-nm (5521ng/g tissue) and 250nm (67ng/g tissue) AuNPs occurred in the testes at 1day postinjection versus the control. However, no significant accumulation of 50- or 100-nm AuNPs occurred in the testes at 1day postinjection versus the control. Although the percentage of the injected dose that accumulated in the testes is relatively low (0.2%), this low percentage represents a very large number of AuNPs. This suggests that AuNPs biodistribute to the testes in a size-dependent manner. In this same study, the 10-nm AuNPs demonstrated the most widespread biodistribution to various organ systems including the blood, liver, spleen, lungs, kidneys, testis, thymus, heart and brain. In addition, 10-, 50-, 100- or 250-nm Au particles all accumulated to the highest extent in the liver and spleen. However, because inductively coupled plasma is a type of plasma source in which the energy is supplied by electric currents that are produced by electromagnetic induction, that is, by time-varying magnetic fields [101] , the quantification of metals in tissue samples using inductively coupled plasma-mass spectrometry may demonstrate that tiny gold particles which are much smaller than nanoparticles are leached from the nano-Au.
Magnetic NPs

The use of magnetic NPs utilizes not only their low toxicity but also their unique magnetic properties, both of which have led to applications in the areas of cell labeling, cell targeting, drug delivery systems and gene delivery. With regard to the low toxicity of magnetic NPs, magnetite NPs coated with polyvinylalcohol (Fe3O4-PVA) spontaneously bind to the sperm cell membrane and penetrate sperm cells without affecting sperm motility or the Ca 2+ -induced or EGF-induced acrosomal reaction [36] .
www.futuremedicine.com

581

Review

Lan & Yang

In order to investigate the toxicity and tissue distribution of magnetic NPs, Kim etal. studied mice injected intraperitoneally with a single 0-, 10-, 25-, 50- or 100-mg/kg body weight dose of 50-nm silica-coated magnetic NPs containing rhodamineB isothiocyanate (MNPs@SiO2RITC) and sacrificed at 1, 2, 3 and 4 weeks postinjection [37] . They found a significant accumulation of MNPs@ SiO2RITC in the testes at 1week and 2weeks postinjection versus the control, indicating that the MNPs@SiO2RITC crossed the BTB. In this same study, MNPs@SiO2RITC also accumulated in the brain at 1, 2, 3 and 4weeks postinjection, indicating that the MNPs@ SiO2RITC may have penetrated the blood brain barrier (BBB) to gain access. The BBB was not damaged by the MNPs@SiO2RITC, since the barrier was still able to exclude Evan blue dye. However, MNPs@SiO2RITC may have gained access to the brain by entering through areas (e.g., circumventricular areas) that are not protected by the BBB. Regarding MNPs@SiO 2 RITC toxicity, body weight, organ weight, serum analysis, blood chemistry, histopathology examination and mutagenicity showed no differences between the MNPs@ SiO2RITC mice and controls. However, rhodamineB isothiocyanate may have leached from the magnetic NPs, so conclusions regarding biodistribution and BTB penetration should be taken with caution. Kwon etal. studied mice that inhaled highefficiency particulate filtered air (control), a low dose (4.9105 particles/cm 3) or a high dose (9.3105 particles/cm3) of 50-nm fluorescent magnetic NPs for 4weeks (4h/day, 5days/ week) [38] . They reported a significant accumulation of 50-nm fluorescent magnetic NPs specifically in the interstitial cells (Leydig cells) of the testes, not the seminiferous tubules. In addition, 50-nm fluorescent magnetic NPs were localized in the brain. They postulated that the magnetic NPs may have gained access to the brain by entering through areas (e.g., circumventricular areas) that are not protected by a BBB or by translocating along the olfactory nerve to the olfactory bulb.
Nano-chitosan

surrounding adjacent tissue 1day after intravenous injection, probably due to NP extravasation through highly permeable tumor vasculature [39] . In addition, a palmitoyl glycol chitosan hydrogel released fluorescein isothiocyanatedextran in a controlled fashion based upon the degree of hydrophobicity, in that a low-hydrophobic chitosan hydrogel released more fluorescein isothiocyanatedextran than a high-hydrophobic chitosan hydrogel [40] . Regarding the delivery of chemotherapeutic drugs into tumors, 300-nm glycol chitosan NPs conjugated to doxorubicin injected intravenously into tumor-bearing mice exhibited comparable anti-tumor activity to free doxorubicin, but with a lower toxicity [41] . However, glycol chitosan NPs conjugated to cisplatin injected intravenously into tumor-bearing mice showed higher anti-tumor activity and lower toxicity compared with free cisplatin [42] . Both of these studies indicate that glycol chitosan NPs conjugated to a chemotherapeutic drug most likely elicit their tumor-homing properties and anti-tumor activity due to the enhanced permeability of the tumor vasculature and enhanced retention of these NPs within thetumor. Choi etal. studied mice that were instilled intratracheally with a single 2-mg/kg body weight dose of 350-nm glycol chitosan NPs labeled with near-infrared fluorescent Cy5.5 and sacrificed at 1 and 6h, and 1, 3, 7 and 14days [43] . They reported a widespread distribution to extrapulmonary organs through the vasculature, such that a significant accumulation of glycol chitosan NPs occurred in the liver and kidney from 1h to 7days; in the spleen from 6h to 7days; in the brain from 1h to 1day; and in the interstitium of the testes from 1h to 7days. The cellular level of nano-chitosan is discussed in Nano-chitosan below.
Nano-carbon

Glycol chitosan NPs show promise as a selective delivery system to shuttle radionuclide imaging probes or chemotherapeutic drugs into tumors. By combining different hydrophobic moieties and hydrophilic polymer backbones, glycol chitosan NPs clearly delineated tumor tissue from
582
Nanomedicine (2012) 7 (4)

Carbon nanotubes consist of carbon atoms arranged in a series of condensed benzene rings structured in tubular form. They can be classified into two general categories: single-walled carbon nanotubes, which consist of one layer of cylinder graphene; and multiwalled carbon nanotubes, which consist of multiple concentric graphene sheets. The physiochemical properties of carbon nanotubes make them a unique nanomaterial with many diverse applications such as vaccine delivery, drug delivery, gene therapy vectors and growth substrates for tissue regeneration [44] . For example, single-walled carbon nanotubes functionalized with polyethylene glycol bound to arginineglycineaspartic acid
future science group

How nanoparticles affect spermatogenesis

Review

(binds to integrin) specifically accumulated in integrin-positive mouse tumors as measured by PET and Raman spectroscopy [45] . In addition, single-walled carbon nanotubes functionalized with prodrug modules specifically targeted tumors [46] . Finally, carbon nanotubes positioned adjacent to bone promoted bone regeneration [47] . In order to study the effects of carbon NPs on the male reproductive system, Yoshida etal. studied mice instilled intratracheally with a 0.1-mg/ mouse dose (ten-times every week for 10weeks) of 14-, 56- and 95-nm carbon NPs or 0.1ml of saline (control group) [48] . They reported no change in body weight, testes weight or epididymis weight in the carbon NP-treated groups versus the control. However, they concluded that carbon NP exposure causes adverse effects on the male reproductive system that are dependent not only on NP size but also NP number, based on histological observations. Bai etal. studied mice injected intravenously with a 5-mg/kg body weight dose (either a single dose or five doses over 13days) of 2030nm amine-functionalized or carboxylate-functionalized carbon NPs or saline (control group) [49] . They found no change in body weight, testes weight or epididymis weight in the carbon treated groups versus the control at 15, 60 or 90days post-treatment. They found that 41, 61 and 151ng of carbon NPs accumulated in the testes at 10min, 60min and 24h post-treatment, respectively. In addition, after treatment with carboxylate-functionalized carbon NPs, the testes showed increased levels of MDA after 15days compared with controls, but returned to normal levels after 60 and 90days. Since MDA is an index of antioxidant status, this may mean that a repair mechanism exists in the testes. The copulation, fertility, gestation and viability indices, along with the average number of live pups per pregnant female in the carbon NP treated groups, showed no difference compared with controls, suggesting that carbon NPs do not affect male fertility. The cellular level of nano-chitosan is discussed in Nano-carbon below.
Nano-Ag

widespread use, the toxicity of nano-Ag in biological and ecological systems has been called into question. In this regard, recent reports indicate that nano-Ag damages neurons [51] , hepatocytes [52] and stem cells [53] . Kim etal. studied rats administered orally with a 30, 125 and 500mg/kg body weight/day dose, or vehicle (control) of 56-nm nano-Ag particles for 13weeks [54] . They found that a significant decrease in body weight occurred at 10weeks in the 125mg/kg body weight/day dose in male treated rats, while a more prominent decrease in body weight also occurred at 4, 5 and 7weeks in the 500mg/kg body weight/day dose male treated rats versus controls. The relative testis weights (only the left testis) showed a significant increase at 13weeks in the 500mg/kg body weight/day dose male treated rats. In addition, nano-Ag significantly accumulated in the testes after 13weeks in all three dose groups compared with controls. Serum cholesterol levels increased significantly at 13weeks in both the 125 and 500mg/kg body weight/day dose male treated rats versus controls, indicating some hepatocyte toxicity. Park etal. studied mice administered orally with a 1-mg/kg body weight dose of 22-, 42-, 71and 323-nm nano-Ag particles or vehicle (controls) for 2weeks [23] . Using this regime, they found that no change in body weight occurred at 2weeks in the 22-, 42-, 71- and 323-nm nanoAg-particle treated mice versus controls. In addition, only 22- and 42-nm nano-Ag particles significantly accumulated in the testes after 2weeks compared with controls. In the same study, Park etal. studied mice administered orally with a 0.25-, 0.5- and 1-mg/kg body weight dose of 42-nm nano-Ag particles or vehicle (controls) for 4weeks. They observed that both alkaline phosphatase and aspartate transaminase serum levels increased in the 1mg/kg body weight dose treated mice versus controls.
Nano-TiO2

If used in appropriate amounts, silver metal and silver dressings do not demonstrate any negative effects on humans [50] . However, the antibacterial activity of silver makes the widespread use of nano-Ag in many commercial products (e.g., shampoo, food packaging, water filters, and medical devices) very attractive. Due to this
future science group

The use of nano-TiO2 utilizes its photocatalytic, anti-UV light, and photoelectric properties, which have led to its application in consumer products such as toothpastes, sunscreens, cosmetics and surface coatings [5558] . In addition, the anatase form of TiO2 induces oxidative DNA damage, lipid peroxidation, increased hydrogen peroxide production and increased nitric oxide production in a human bronchial epithelial cell line [59] . In order to explore the toxicity of nano-TiO2 in male offspring, Takeda etal. studied pregnant
www.futuremedicine.com

583

Review

Lan & Yang

mice injected subcutaneously with a 100-ng dose of 2570nm TiO2 or saline (control) at 3, 7, 10 and 14days postcoitum [60] . They reported that nano-TiO2 accumulated in the brain and testes of male offspring from nano-TiO2-treated pregnant mothers. Regarding the brain, nano-TiO2 was found in the olfactory bulb and frontal and temporal lobes of the cerebral cortex. In addition, many apoptotic cells were found in the olfactorybulb. The cellular level of nano-TiO2 is discussed in Nano-TiO2 below.
C60 fullerene

Nano-SiO2

A fullerene is a graphite-like structure that consists of stacked graphene sheets of linked hexagonal, pentagonal, or heptagonal rings. A fullerene is composed of entirely of carbon and may exist in the form of a hollow sphere (also called a buckyball), ellipsoid or tube (also called a carbon nanotube or buckytube). C60 fullerene (also called buckminsterfullerene) was the first fullerene discovered and resembles a geodesic dome. The various known fullerenes demonstrate the potential for usefulness in various technological applications, especially in material science, electronics and nanotechnology. In order to investigate whether hydrated C60 fullerene (C 60HyFn) could prevent testicular dysfunction induced by streptozotocin-induced TypeI diabetes, Bal etal. studied rats divided into four groups: diabetic rats; C60HyFn-treated diabetic rats; C60HyFn-treated control (nondiabetic) rats; and control (nondiabetic) rats [61] . As soon as hyperglycemia was detected in the streptozotocininjected rats, the rats were orally administered (via their drinking water) with a 4-g/kg body weight dose of C60HyFn for 5weeks. They reported that the relative weights of the right caudal epididymis, seminal vesicles, prostate gland and sperm motility and epididymal sperm concentration significantly decreased in diabetic rats versus control (nondiabetic rats). However, the relative weights of all of the above structures showed no difference in the C60HyFn-treated diabetic rats versus control (nondiabetic) rats which indicates a protective effect of C60HyFn against testicular dysfunction caused by diabetes. In addition, C60HyFn protected against the following diabetes-induced testicular dysfunctions: histopathological changes in the testes; low serum testosterone; reduced testicular glutathione; low serum tocopherol; and high lipid peroxidation. Since these findings conflict with other previously reported studies [62,63] , more studies on the protective effect of C60HyFn should be performed.
584
Nanomedicine (2012) 7 (4)

SiO2 (also called silica) is an oxide of silicon most noted for its hardness and commonly found in nature as sand or quartz. Nano-SiO2 particles are typically 5100nm in diameter, with a high surface area in the range of 2550m2/g. The uses of nano-SiO2 include solar cell applications, high temperature insulators, gas sensors, surface coatings, and additives to rubber and textile products. Nano-SiO2 is a recognized environmental hazard due to its involvement in much respiratory pathology. However, the impact of nano-SiO2 on the reproductive system remains unanswered. Lin etal. studied rats that were instilled intratracheally with either a 1.5- or 7.5-mg/kg body weight dose of 2040-nm SiO2 NPs or 110m microparticles once every 2days for 5weeks [64] . They found that body weight was significantly reduced in the 7.5-mg/kg body weight dose group versus the control group. However, the relative organ weights showed no differences. Regarding oxidative damage in the testes, MDA, SOD and GSH-Px activity levels were measured. They reported that testis MDA levels in the 1.5(both NPs and microparticles) and 7.5-mg/kg body weight dose groups (NPs only) significantly increased versus the control group. The testis SOD levels in the 7.5-mg/kg body weight dose group (NPs only) significantly decreased versus the control group. The testis GSH-Px levels in 7.5-mg/kg body weight dose group (both NPs and microparticles) also significantly decreased versus the control group. The serum MDA activity level in the 7.5-mg/kg body weight dose group (NPs only) significantly increased versus the control group. These results indicate that the SiO2 NPs cause a higher toxicity when compared with SiO2 microparticles at the same exposure level, possibly due to lipid peroxidation in thetestes. Fan etal. studied rats that were exposed to air in inhalation chambers containing either a 100 or 300mg/m3 air dose of 10nm SiO2 NPs or 15-m SiO2 microparticles for 2h every other day for 65days [65] . They reported that the body weight was significantly lowered in the 100mg/m3 of air dose group (NPs only) and the 300mg/m3 of air dose group (NPs only) versus the control group. The relative testes weight was significantly decreased in the 100mg/m3 of air dose group (NPs only) versus the control group. However, the relative epididymis weight was significantly increased in the 100mg/m3 of air dose group (NPs only) and the 300mg/m3 of air dose group (NPs only) versus the control group. In regards to oxidative damage in the testes,
future science group

How nanoparticles affect spermatogenesis

Review

MDA, SOD, lactate dehydrogenase-C4 and succinate dehydrogenase activity levels were measured. They found that MDA, SOD, lactate dehydrogenase-C4 and succinate dehydrogenase activity levels were all significantly increased in the 100mg/m3 of air dose group (microparticles only) and the 300mg/m3 of air dose group (NPs and microparticles) versus the control group. The serum SOD activity level in the 100mg/m3 of air dose group (NPs only) and the 300mg/m3 of air dose group (NPs and microparticles) significantly increased versus the control group. The cellular level of nano-SiO2 is discussed in Nano-SiO2 below.
Summary

NP exposure and spermatogenesis perturbations remain scarce. In this section we will highlight studies that offer essential data on the testes and related cells (e.g., Sertoli cells, Leydig cells and spermatogenic cells).
Nano-Se

The main results (discussed in Clinical level) of the abovementioned journal articles are summarized in Table1. The indices presented in Table1 are the common indices studied in the journal articles reviewed above. Table1 shows the gaps in our knowledge concerning the relationship between NPs and spermatogenesis because most studies are performed from a toxicology or animal husbandry viewpoint. In this regard, the experimental design used by Bai etal. provides a good model for studies directly focusing on the effect of NPs on spermatogenesis [49] . Taking a broad view of the abovementioned journal articles, it becomes clear that indices such as route of administration, exposure time, and surface modification of the NPs all play a serious role in the experimental outcome, and should be taken into account in all future experiments. In addition, the broad view illustrates that although many different indices were measured in the abovementioned journal articles, the exact cellular or molecular mechanisms were not explored. Therefore, many of the measured indices may be a superficial indication of perturbations at deeper cellular or molecular levels. We suggest that future experiments begin to focus on cellular and molecular mechanisms to deduce the basic NP toxicology on spermatogenesis.
Cellular level

Shi etal. studied Boer goats fed diets containing nano-Se at a Se concentration of 0.3mg/kg body weight (Se-supplemented group) or 0mg/kg (Se-deficient control group) for 12weeks [20] . They found that in the Se-supplemented group the sperm ultrastructure had the following characteristics: a generally normal appearance; the nuclear envelope showed integrity; the mitochondria showed a tight array (no gaps) in the helix around the mid-piece; and the mitochondria showed a normal oblong-shape and smooth intact mitochondrial membranes. However, in the Se-deficient group the sperm ultrastructure had the following characteristics: a generally abnormal appearance; the nuclear envelope showed damage; the mitochondria showed gaps in the helix around the mid-piece; and the mitochondria showed abnormal shapes and extensive vacuolation. This study indicates that nano-Se reduced the sperm abnormalities and therefore may be used to raise sperm production in Se-deficient regions.
Nano-chitosan

Choi etal. studied the biodistribution of nanochitosan (details of the experimental design have been previously explained) using 350-nm glycol chitosan NPs labeled with near-infrared fluorescent Cy5.5 and confocal laser scanning microscropy [43] . They observed chitosan NPs only within the interstitium of the testes, suggesting that nano-chitosan does not penetrate the BTB, the interstitium of the prostate gland, and the interstitium of the seminal vesicle.
Nano-carbon

All the studies highlighted in the Clinical level section of this review indicate that NPs are found in the testes and penetrate the BTB, except in some special cases due to size dependence or nano-chitosan. Because most of these studies were directed towards toxicology or animal husbandry issues, the status of the testes was not specifically explored, except in some studies where sperm quality was measured. Consequently, studies that explore a direct relationship between
future science group

Yoshida etal. studied mice instilled intratracheally with 14-, 56- and 95-nm carbon NPs (details of the experimental design have been explained previously) and explored the effects on the testes using light microscopy [48] . They histologically observed both vacuolation and degenerative changes in the seminiferous tubules in the carbon NP-treated group versus the control group. In addition, partial vacuolation within the Sertoli cells and Leydig cell dysfunction (causing increased testosterone production) occurred. Finally, they observed decreased daily sperm production in the carbon NP-treated mice versus the control.
www.futuremedicine.com

585

Review

Lan & Yang

Table1. Summary of the effects of nanoparticles on spermatogenesis on all three levels (clinical, cellular and molecular).
Nanoparticle Features of material
Selenium (Se) Normal Normal

Size (nm)
6080 6080

Test animals (age)

Boer goat (2 month) Black taihang goat (90 3days) SpragueDawley rats (4weeks)

Dose

Administration Time after administration or test time

Oral Oral 12weeks 90days

0.3mg/kg every day 2500mg/animal every day

Normal

2060

0.22, 0.31, 0.42mg/kg body weight per day

Oral

13weeks

Magnetic (Fe3O4)

Within a silica shell Fluorescent

50 50

ICR mice (6weeks) ICR mice (5weeks) ICR mice (5weeks)

100, 50 and 25mg/kg 4.891052.37104 / cm3 and 9.34105 5.11104 /cm3 0.1 mg/mouse and 1.46g/mouse, both ten-times per week Scheme 1: a single dose of 5mg/kg Scheme 2: five doses over 13days at 5mg/kg 30, 125, 500mg/kg

Intraperitoneally Air

4 weeks 4weeks (4h/day 5day/ week)

Carbon (C)

Nanotube

14, 56, 95

Intratracheally

Silver (Ag)

Amine (NH2) and carboxylate (COOH) Normal

2030

Adult male BALB/c mice

Intratracheally

90days

56

F344 rats (5weeks)

Oral

90days

Normal Titanium dioxide (TiO2) Normal

22, 42, 71, 232 2570

ICR mice (6weeks) Pregnant Slc:ICR mice

1mg/kg 100l of 1mg/ml at 3, 7, 10 and 14days postcoitum

Oral Subcutaneous injected

14days Offspring at 4days or 6weeks

Fullerene (C60 ) C60HyFn

Rat

4g/kg daily for 5weeks

Oral

5weeks

Silicon dioxide Normal (SiO2) Normal

1020

Wistar rats

7.5mg/kg (high group) and Intratracheally 1.5mg/kg (low group) 100mg/kg (low) and 300mg/kg (high) Air

5weeks

105

Wistar rats

65days

C60HyFn: C60 hydrated fullerene; GSH: Glutathione; ICR: Imprinting control region; LDH: Lactate dehydrogenase; MDA: Malondialdehyde; MWCNT: Multi-walled carbon nanotube; NA: Not affected; NM: Not mentioned; Px: Peroxidase; SDH: Succinate dehydrogenase; SH: Significantly higher than the control; SL: Significantly lower than the control; SOD: Super oxide dismutase.

586

Nanomedicine (2012) 7 (4)

future science group

How nanoparticles affect spermatogenesis

Review

Body weight
NM SH

Relative Oxidative Distribution reproductive stress indicator features and organ (testis) (testis) detection
NM NM GSH-Px (SH) NM Detected Detected

Seminiferous tubule damage

Sperm damage

Locations of Ref. nanoparticles

[20]

[22]

NA

NA

NM

Detected

NM

The control: the membranes enveloping the elongated nucleus of spermatozoon and mitochondria are abnormal

NM

[29]

NA NM

NM NM

NM NM

Detected Detected

[37] [38]

NA

NA

NM

Detected

NA

NA

High dose: 4, 5, 7weeks (SL); low dose: 10weeks (SL) NM SL

High dose: left testis 90days (SH) NA NA

MDA of 15days, MWCNT-COOH Scheme 2 group (SH) NM

Detected; a repairing mechanism found NM

Nanotreated groups: the rate is higher than the control Higher in the 15th day; returns to normal after 60days

NM

NM

[48]

NA

NM

[49]

[54]

NM NM

Detected; size dependence Detected

[23]

The control: the number of Sertoli cells is decreased and the damaged Sertoli cell mitochondria numbers are significantly higher

NA

Spermatids, Sertoli cells and Leydig cells

[60]

NM

NA

High group (SL)

NA

MDA (NA) GSH Detected level has a restore mechanism MDA high and Detected low (SH) GSH low (SH) High: LDH-C4, SDH, MDA and SOD (SH) Detected NM The nano-treated NM groups: the mobility and normality of the sperm are higher

[61]

[63]

Both high and Low (SL) low groups (SL)

[65]

future science group

www.futuremedicine.com

587

Review

Lan & Yang

Bai etal. studied mice injected intravenously with 2030-nm carbon NPs (details of the experimental design have been explained previously) and detailed the effects on the testes observed by light microscopy [49] . The testes showed little or no histological changes after a single-dose treatment. However, they observed partially damaged seminiferous tubules, a reduced thickness of the germinative layer, and a reduced number of spermatogonia in the mice treated with five doses of carboxylated carbon NPs after 15days. In addition, partial disappearance and vacuolization occurred within the Sertoli cells. The abovementioned histological disturbances were observed only occasionally after 60 and 90days, indicating that reversal of these disturbances may occur.
Nano-TiO2

Takeda etal. studied pregnant mice injected subcutaneously with 2570nm nano-TiO2 in order to explore the biodistribution of nano-TiO2 in male offspring by energy-dispersive x-ray spectroscopy [60] (details of the experimental design have been previously explained). They observed nano-TiO2 within spermatids, Sertoli cells and Leydig cells of the male offspring. In addition, the seminiferous tubules showed disorganization while the daily sperm production, sperm motility and Sertoli cell number all significantly decreased. This study remains difficult to compare to other studies, not only due to diverse NP characteristics but also because the pregnant mice were exposed to nano-TiO2 and the effects were measured on the male offspring. In this regard, nano-TiO2 penetrates the placental membrane and affects the male reproductive system during embryological development.
Nano-SiO2

cellular level suggest that NPs elicit negative effects on male reproductive cells (e.g., Sertoli cells, Leydig cells, spermatogonia, spermatocytes, spermatids and spermatozoa) based on histological observations. However, these same studies at the clinical level in general show that NPs do not elicit any negative effects based on clinical measurements. For example, Yoshida etal. observed that nano-carbon caused both vacuolation and degenerative changes in the seminiferous tubules and partial vacuolation within Sertoli cells and Leydig cell dysfunction [48] . However, they reported that nano-carbon caused no change in body weight, testes weight or epididymis weight. Due to the ongoing interest in finding the mechanism(s) of NP action on spermatogenesis, understanding the clinical and cellular changes in the testes due to NP exposure plays an important first step, but clearly we need studies that explore details at the molecular level. For example, some NPs are delivered into the sperm and affect protein transport because microtubules are damaged. However, looking at the situation from a different perspective, perhaps NPs directly affect gene expression first, then protein transport is diminished, and finally microtubule damage can be observed.
Molecular level

Fan etal. studied rats that were exposed to air containing 100 or 300mg/m3 air dose of 10-nm SiO2 NPs or 15m SiO2 microparticles [65] (details of the experimental design have been previously explained) and detailed the effects on the testes by light microscopy. They found histopathological changes in the testes, reduced sperm counts and reduced sperm motility due to SiO2 NP exposure. These authors also concluded that SiO2 NPs cause a higher toxicity when compared with SiO2 microparticles at the same exposure level.
Summary & discussion

The main results (discussed in Cellular level) of the abovementioned journal articles are summarized in Table1. In general, the studies at the
588
Nanomedicine (2012) 7 (4)

Many studies at the molecular level aim to detect the effect of NPs on gene expression and protein expression levels of various proteins specific to the tissue under study. In this regard, quantitative real-time PCR and western blot analysis have been applied most often in toxicological studies involving the liver, spleen and lung. However, studies using these molecular techniques aimed at detecting the effect of NPs on gene and protein expression levels for various proteins specific to the testes and/or spermatogenesis are just now becoming routine. Ramdhan etal. studied rats exposed to air containing a low concentration (15.41.0g/m3 mass weight), medium concentration (36.41.2g/m3 mass weight), high concentration (168.82.7g/m 3 mass weight) and zero (control) nanomaterial-rich diesel exhaust (NR-DE) for 1and 2months (5h/day, 5days/week) [66] . They reported that plasma testosterone levels increased in the low and medium groups after 1and 2months of exposure to NR-DE. In order to explore the molecular mechanisms causing the increased plasma testosterone levels, they used quantitative real-time PCR and western blot analysis to measure gene and protein expression levels of proteins related to testosterone production in
future science group

How nanoparticles affect spermatogenesis

Review

the testes. They found that the StAR protein, cytochrome P450 side chain cleavage, growth hormone receptor mRNA and protein expression levels increased in the low and medium groups after NR-DE exposure. In addition, IGF-1 mRNA expression levels increased in the low group after NR-DE exposure. Consequently, the disruption of testosterone biosynthesis caused by NR-DE exposure may be explained by molecular changes in StAR and P450 side chain cleavage expression levels via growth hormone signaling. This study is an excellent model whereby the clinical finding of increased testosterone levels was explored further to the molecular level to ascertain a possible mechanism. Invitro studies The number of invitro studies involving the effect of NPs on spermatogenesis or the male reproductive system in general is limited. This is somewhat disconcerting, since invitro studies are better suited to explore molecular mechanism of action. Invitro studies provide fewer variables that may complicate data interpretation than invivo studies and can target specific cell types (e.g., Leydig cells, Sertoli cells and so on).
Leydig cells

Acrosome Equator region Midpiece

Zona pellucida

Egg membrane

Nanomedicine Future Science Group (2012)

Figure1. The outline of a mammalian sperm and the process of fertilization. (A)The locations of the three important parts of spermatozoa. (B)The first recognition with the zona pellucida is achieved by the acrosome (left) and the second recognition with the egg membrane is achieved by the equator region (right).

The Leydig cells secrete testosterone, an essential hormone for spermatogenesis. Consequently, Leydig cells provide an excellent target cell to study the invitro effects of NPs on the male reproductive system. Komatsu etal. studied the mouse Leydig cell line TM3 exposed to 2570-nm TiO2, diesel exhaust particles (DEP), and 14nm CB at 1, 10, 100 and 1000g/ml and 0 (control) concentrations for various time periods [67] . Using electron microscopy, they observed that all three NPs were present within the TM3 cells as random cytoplasmic agglomerates not associated with any particular organelle, and were not found in the nucleus. Based on the images and illustrations of the study by Komatsu etal. [67] , we produced Figure1 to show the interactions between Ledig cell and NPs. They reported that TM3 cells exposed to 100-g/ml TiO2 NPs for 48h showed a severe reduction in cell viability, whereas TM3 cells exposed to 100g/ml of DEP or CB showed no reduction in cell viability. In addition, they reported that TM3 cells exposed to 100-g/ml TiO2 NPs and 100-g/ml DEP for 24h showed a decreased cell proliferation that was restored by 48h. In order to investigate molecular mechanisms associated with these changes, they studied the gene expression levels of HO-1 in TM3 cells,
future science group

which is a sensitive marker for oxidative stress and StAR, which is an important protein in testosterone biosynthesis. They found that TM3 cells exposed to 30 and 100g/ml of DEP for 16h resulted in a significant increase in HO-1 gene expression versus the controls. In addition, TM3 cells exposed to 30g/ml DEP, 10 or 30g/ml CB for 48h showed a significant increase in StAR gene expression versus the controls. This study indicates the usefulness of TM3 invitro studies to investigate the biotoxicity of various nanoparticles.
Germline stem cells

Braydich-Stolle etal. studied C18-4 germline stem cells (established from mouse Type A spermatogonia) exposed to 15-nm silver NPs (nanoAg), 30-nm molybdenum NPs (nano-Mo), and 30-nm aluminum NPs (nano-Al) at 5, 10, 25, 50 and 100g/ml, and 0 (control) concentrations for 48h [53] . Cadmium oxide (1000nm diameter) was used as a positive control due to its known cytotoxic properties. Using phase contrast microscopy they observed that C18-4 cells exposed to 10-g/ml nano-Ag showed signs of necrosis and apoptosis, C18-4 cells exposed to nano-Mo showed no changes, and C18-4 cells exposed to nano-Al showed signs of accumulation of nano-Al particles in the cytoplasm but were unable to enter the nucleus. In addition, measured by the MTS assay, C18-4 cells exposed to 10-g/ml nano-Ag, 50 or 100g/ml nanoMo demonstrated significantly reduced mitochondrial function (and, by extension, reduced
www.futuremedicine.com

589

Review

Lan & Yang

cell viability). C18-4 cells exposed to 5-g/ml nano-Ag, 5100-g/ml nano-Mo and 5100g/ml nano-Al showed significantly increased lactate dehydrogenase leakage. Finally, C18-4 cells exposed to 5-g/ml nano-Ag, 50-g/ml nano-Mo and 510-g/ml nano-Al showed a significantly increased number of apoptotic cells as measured by the Vybrant Apoptosis Assay Kit (Invitrogen, CA, USA). This study stands as a good example of an invitro study exploring NP toxicity in the male reproductive system, although data on possible molecular mechanisms behind these observations are lacking.
Spermatozoa

Makhluf etal. studied bovine sperm exposed to magnetite coated with polyvinyl alcohol (Fe3O4-PVA) at 7.36mM or 0 (control) concentrations of iron (Fe) ions for various time periods [36] . They observed that sperm took up Fe3O4 PVA quickly in the first 20min of incubation, probably owing to it binding to the sperm cell membrane. This was followed by a partial release of Fe3O4-PVA from the sperm cell membrane, Fe3O4-PVA particles were then taken up linearly into the sperm cell cytoplasm at 50140min after incubation. The Fe3O4-PVA in the sperm cell cytoplasm remained both free and bound to the acrosome and mitochondria, respectively. Based on the images and illustrations of the study of Makhluf etal. [36] we produced Figure2 to show

Lipid droplet

Nucleus Phagosome

Mitochondria

the interactions between spermatozoa and NPs. In addition, sperm cells exposed to Fe3O4-PVA for 4h, A23187 (Ca2+ ionophore), and EGF show a normal acrosomal reaction, indicating that Fe3O4PVA exposure does not compromise this important sperm function. Finally, sperm cell motility was not affected by Fe3O4-PVA incubation. Wiwanitkit etal. studied human sperm exposed to AuNPs at 44ppm or 0 (control) concentrations for 15min [68] . They found that human sperm exposed to nano-Au showed fragmentation and clumping as observed by light microscopy. In addition, 75% of human sperm cells exposed to nano-Au were motile whereas 95% of control human sperm cells were motile (that is, a 20% reduction is sperm motility). The study of NP toxicity directly on mature sperm cells is a fairly practical method to ascertain whether the fertilization capability of sperm will be impacted. The process of fertilization comprises a number of steps, which include: sperm capacitation; spermzona pellucida binding; the acrosome reaction; penetration of the zona pellucida; spermoocyte membrane binding; egg activation and the cortical reaction; and the zona reaction. Consequently, many steps exist where NPs may exert their toxic effect on the fertilization capability of sperm. The study by Makhluf etal. only explored the acrosome reaction, so although some indication of fertilization capability of sperm was established in this study, many other steps were left unaddressed [27] . Thus, NPs effects on the three parts of sperm: the acrosome, which is responsible for the acrosome reaction; the equatorial segment, which is responsible for the spermoocyte membrane binding; and the midpiece, which is responsible for the vigor of sperm, all of which should be fully studied. Figure3 shows a simplified picture of fertilization, emphasizing the importance of these steps in the process of fertilization.

NP (TiO2, DEP, CB) Ledig cell

Nanomedicine Future Science Group (2012)

Figure2. The interaction between Ledig cells and nanoparticles (titanium dioxide, diesel exhaust particle and carbon black). All three NPs (TiO2, DEP and CB) were present within the Ledig cells as random cytoplasmic agglomerates not associated with any particular organelle and were not found in the nucleus [67] . CB: Carbon black; DEP: Diesel exhaust particle; NP: Nanoparticle; TiO2: Titanium dioxide.

How do NPs penetrate the BTB? The BTB consists predominantly but not solely of tight junctions (zonula occludens) between neighboring Sertoli cells but also of actin-based adherens junctions and possibly intermediate filament-based desmosome junctions. The BTB creates a basal compartment and an adluminal compartment that allow for a postmeiotic environment appropriate for germ cell development and protect germ cells from toxins (e.g., NPs), infections and immune responses in order to maintain male fertility [69] . However, the BTB undergoes extensive restructuring during the process of germ cell migration from the basal
future science group

590

Nanomedicine (2012) 7 (4)

How nanoparticles affect spermatogenesis

Review

compartment to the adluminal compartment. In this regard, BTB restructuring probably involves TGF b3 via the TGF b3/MEKKs/p38MAP kinase signal transduction pathway [70] . BTB restructuring also probably involves TNF a via the TNF a /ILK/p130Crk/MAP kinase signal transduction pathway [71] . In addition, the mechanical aspects of the BTB have been somewhat simplistically implicated as the sole player in making the testes an immunoprivileged site [72] . However, we now know that Sertoli cells produce anti-inflammatory cytokines under the influence of adjacent germ cells and residual bodies that include IL1a, IL6 and activinA [73] . These cytokines directly modulate the development of spermatogonia and spermatocytes; the Sertoli cell response to follicle-stimulating hormone; and the integrity of the BTB. In addition, testosterone and other androgens orchestrate an anti-inflammatory effect (by the inhibition of proinflammatory cytokines), which shifts the cytokine balance towards a more immunoprivileged site and perhaps strengthens the integrity of the BTB [74] . Consequently, what this means regarding NP toxicity is that NP exposure may produce a generalized inflammation in the host and effect Leydig cells, causing reduced testosterone serum levels which then weaken the integrity of the BTB. So, NP toxicity on postmeiotic spermatogenic cells may not be a simple case of NPs percolating through the BTB based on size or specific surface modifications. In summary, the elucidation of the underlying molecular mechanism involved in the toxicity of individual NPs will require a painstaking effort that investigates both direct and indirect effects onBTB. In this regard, Park etal. studied mice administered orally with a 1-mg/kg body weight dose of 22, 42, 71 (small-sized NPs) and 323-nm (largesized NPs) nano-Ag particles or vehicle (control) for 2weeks [23] . They found that all small-sized nano-Ag particles caused a bioaccumulation of Ag within the testes (i.e., NPs possibly penetrated the BTB), whereas large-sized nano-Ag particles did not. Interestingly, TGFb serum levels significantly increased in the small-sized nano-Ag particle treated mice versus the large-sized nanoAg particle treated mice or controls. Finally, TGFb, cytokine serum levels (e.g., IL1, IL4, IL6, IL10 and IL12), B-lymphocyte distribution and IgE production significantly increased in a dose-dependent manner when repeated doses of small-sized nano-Ag particles at different concentrations were administered to mice versus controls. These results strongly suggest that nano-Ag particle penetration of the BTB
future science group

The magnetic NPs (gray shading) penetrate the sperm and bind to the acrosome The magnetic NPs (gray shading) bind to the sperm cell membrane The magnetic NPs (gray shading) penetrate the sperm and bind to the mitochondria

Nanomedicine Future Science Group (2012)

Figure3. The interaction between sperm atozoa and magnetic nanoparticles [36] . NP: Nanoparticle.

may not be solely a matter of particle size but may also involve the production of an inflammatory response that weakens the integrity of the BTB. However, the 71-nm treated group, although with an inflammatory response, showed that 71nm nano-Ag particles did not penetrate the BTB, thus a hypothesis named elevator door may reveal the mechanism (Figure 4AB) : after treating with non-NPs, because there is no inflammatory response, the scale of gap of the BTB is not changed. Thus, non-NPs cannot penetrate BTB. The situation can be visualized as being analogous to an unpressed elevator button that leaves the door unopened, keeping an individual outside. Because of the existance of inflammatory responses, the size of the BTB gap becomes larger after treatment with NPs. That is why the smaller NP can penetrate BTB easily; however, it may still keep some larger NPs out. In addition, Meng etal. reported that testosterone may act in concert with some cytokines to strengthen the integrity of the BTB [75] . Warrenetal. [76] and Delfino etal. [77] reported that both testosterone secretion and androgen receptor expression increased in response to cytokines. NPs possess the capacity to penetrate the BTB [78] . In addition, NP may also penetrate barriers in the skin, lung, intestine and brain (i.e., the BBB). The BBB has been one of the most studied barriers because of its implications for human health and the difficulty it causes in delivering drugs to the CNS [7982] . However, the mechanisms involved in NP penetration of the BBB may not be applicable to the BTB, since the BBB
www.futuremedicine.com

591

Review

Lan & Yang

BTB U Ag Sertoli cell X

Spermatozoa BTB

Treated with non-nanoparticles

Sertoli cell

Spermatocyte

Sertoli cell

Sertoli cell X

Sertoli cell

BTB W Ag Treated with nanoparticles Y

Spermatogonium Sertoli cell Tight junction Perspective

V Ag Sertoli cell

Sertoli cell

Nanomedicine Future Science Group (2012)

Figure4. The hypothesis of how nano-Ag penetrates the bloodtestis barrier. (A) The outline of part of the seminiferous tubule. (B)only depicts the Sertoli cells which contact with the basal lamina viewed from the outside of seminiferous tubles to the inside of seminiferous tubles. The elevator door hypothesis: the mathematical relationship among those indices in the figure is U>>V>Y>W>X. After treating with non-nanoparticles, because there is no inflammatory response, the scale of the gap of the BTB remains unchanged. Thus, non-nanoparticles cannot penetrate the BTB. After treatment with nanoparticles, because of the existing inflammatory responses, the scale of the gap of the BTB becomes larger. That is why smaller nanoparticles can penetrate the BTB easily; however, it may still keep some larger nanoparticles out because the size of the larger nanoparticle is larger than the size of the BTB. BTB: Bloodtestis barrier.

studies employed lipophilic or lipid-coated NPs. In addition, the characteristics of the BBB may be quite different to those of the BTB. The BBB components have been traditionally described as: tight junctions (zonula occludens) between endothelial cells; the basement membrane; and the foot processes of astrocytes [83] . However, the BBB is more than a physical barrier consisting of the above-mentioned morphological components. The BBB is also a physiological barrier where many intramembranous ATP-driven drug export pumps have been localized on the luminal (blood) side of brain capillary endothelial cells. The ATP-binding cassette transport protein called P-glycoprotein acts as a critical barrier to the entry of many drugs (e.g., vinca alkaloids, doxorubicin or taxanes) in the CNS by acting as an efflux pump to pump the drug out of the endothelial cells as fast as it can enter [83] . As to the ability of water-soluble agents to penetrate the BBB, Fernandes etal. described a paracellular aqueous pathway [81] . Even though silver, gold and magnetic NPs are water-soluble, they are still unable to penetrate the BBB using the paracellular aqueous pathway because their diameters are too large. Consequently, compounds circulating in the blood can gain entrance into the CNS either by
592
Nanomedicine (2012) 7 (4)

lipid-mediated transport of small molecules or by completely bypassing the BBB at the median eminence, lamina terminalis or area postrema [84] . Interestingly, the testes possess no such bypassareas.

Conclusion & discussion In this review, we approached the topic: How do NPs affect spermatogenesis? In short, our knowledge on the effect of NPs on spermatogenesis and their attendant molecular mechanisms remains in its early stages. The invivo studies at the clinical level provide important information concerning the selection of which NPs demonstrate toxicity and therefore deserve further exploration. In this regard, the studies by Bai etal. [49] and Ramdhan etal. [66] can be regarded as model invivo studies to explore NP toxicity on spermatogenesis. Also, the studies by Komatsu etal. [67] and Braydich-Stolle etal. [53] can be regarded as model invitro studies to supplement invivo studies and to explore molecular mechanisms more efficiently. One of the general takehome conclusions of these studies is that each NP must be studied individually because NPs have disparate effects. Furthermore, NP size, route of administration and other characteristics such as surface structure, chemical properties and dose
future science group

How nanoparticles affect spermatogenesis

Review

all need to be taken into account. Surface structure and chemical properties are always taken into consideration less. Many of the included studies use high doses compared with potential human exposure, so reasonable doses should also be taken into consideration in future studies. Since the molecular mechanisms involved in NP toxicity to spermatogenesis are not wellestablished, the ability to adequately link the data from the clinical and cellular studies remains weak and may generate some less than accurate conclusions and/or speculations. Regarding this, the morphological study of NP toxicity on spermatogenesis may show a specific pathological finding similar to nuclear membrane disruption. However, the NPs may not directly disrupt the nuclear membrane, but instead, directly affect the cytoskeleton and then secondarily disrupt the nuclear membrane. In addition, NPs may be endocytosed by spermatogenic cells and/or Leydig cells, induce cellular changes, and then be exocytosed so that no morphological evidence of NP damage is ever apparent. The abovementioned issues add to the complexity of research studies involving NP toxicity. In order to understand the steadily declining fertility rates in the human population, we need to study not only the causes of reduced sperm quality and number, but also, the sperm/egg recognition process during fertilization. The acrosome, the mitochondrial cloud and the equator region of the sperm all play a vital role in sperm/egg recognition process during fertilization. Consequently, NP toxicity studies should address the molecular mechanisms involved in sperm/egg recognition events. Finally, the question: How do NPs penetrate the BTB? plays a vital role in explaining NP toxicity on spermatogenesis. In comparison with studies involving NP penetration of the BBB, studies involving the BTB are few. Since the molecular mechanisms involved in penetrating the BBB may be entirely different to those involved in penetrating the BTB, extrapolation
Executive summary

of BBB mechanisms to the BTB may be unwarranted, although some clues may point to those such as the elevator hypothesis illustrated in Figure4AB .

Future perspective As NPs are being used in a variety of fields, such as medical applications, there is a growing number of studies on how NPs affect spermatogenesis and male infertility. Future steps should focus on revealing the mechanism of how NPs affect spermatogenesis (e.g., clinical, cellular and molecular levels). Meanwhile, the mechanism of how NPs penetrate the BTB should also be investigated and a detailed mechanism should be revealed based on present studies. Understanding the effects of nanotoxicology on male reproductive organ will be beneficial in understanding the problem of male infertility and setting up plans to solve it. The way to test the elevator hypothesis may need the assistance of confocal laser microscopy and transmission electron microscopy in order to achieve high-quality images of this penetration process.
Acknowledgements
The authors are indebted to the students in the Sperm Laboratory at Zhejiang University for their valuable discussion and inspiring help. The authors appreciation also goes to RDudek (The Brody School of Medicine at East Carolina University, NC, USA), who provided solid and constructive contribution to this manuscript.

Financial & competing interests disclosure

This work was supported by National Natural Science Foundation of China (No 31072198) and the National Basic Research Program of China (973 Program, Grant number: No 2012CB967902). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

How do nanoparticles affect spermatogenesis? Although the toxicological data of how nanoparticles (NPs) affect spermatogenesis is abundant but it is not complete, and more data should be made available. The complete mechanism of how NPs affect spermatogenesis is not yet known. Modern cellular and molecular techniques, should be used to reveal this mechanism. Although spermatogenesis is a very important process for male fertility, the acrosome, equator region and midpiece, which are important for fertilization, should also be carefully studied. How do NPs penetrate the bloodtestis barrier? The mechanism of how NPs penetrate the bloodtestis barrier may be related to the impaired integrity of the bloodtestis barrier induced by inflammatory response, which may in turn be induced by NPs.

future science group

www.futuremedicine.com

593

Review
References

Lan & Yang

16 Farrow S. Falling sperm quality: fact or

30 Ferrari M. Cancer nanotechnology:

Papers of special note have been highlighted as: n of interest nn of considerable interest
1

fiction? BMJ 309(6946), 12 (1994).

17 Hoover MD, Stefaniak AB, Day GA, Geraci

opportunities and challenges. Nat. Rev. Cancer 5(3), 161171 (2005).

31 IE-Sayed I, Huang X, EI-Sayed M. Selective

Buzea C, Pacheco I, Robbie K. Nanomaterials and nanoparticles: sources and toxicity. Biointerphases 2(4), MR17MR71 (2007). Valiev R. Materials science: nanomaterial advantage. Nature 419(6910), 887889 (2002). Aitken RJ, Chaudhry MQ, Boxall ABA, Hull M. Manufacture and use of nanoparticles: current status in the UK and global trends. Occup. Med. (Lond). 56(5), 300306 (2006). Zamborini FP, Bao L, Dasari R. Nanoparticles in measurement science. Anal. Chem. 84(2), 541576 (2011). Delouise LA. Applications of nanotechnology in dermatology. J.Invest. Dermatol. 132(3 Pt 2), 964975 (2012). Lim ZZ, Li JE, Ng CT, Yung LY, Bay BH. Gold nanoparticles in cancer therapy. Acta Pharmacol. Sin. 32(8), 983990 (2011). Lens M. Recent progresses in application of fullerenes in cosmetics. Recent Pat. Biotechnol. 5(2), 6773 (2011). Stern ST, McNeil SE. Nanotechnology safety concerns revisited. Toxicol. Sci. 101(1), 421 (2008). Ema M, Kobayashi N, Naya M, Hanai S, Nakanishi J.Reproductive and developmental toxicity studies of manufactured nanomaterials. Reprod. Toxicol. 30(3), 343352 (2010). Peters S, Hankin S, Stone V. Identification of the mechanisms that drive the toxicity of TiO2 particulates: the contribution of physicochemical characteristics. Part. Fibre Toxicol. 6, 33 (2009).

2 3

CL. Exposure assessment considerations for nanoparticles in the workplace. In: Nanotoxicology: Characterization, Dosing, and Health Effect. Monteiro-Riviere NA, Tran CL (Eds). Informa Healthcare, Zug, Switzerland (2007).
18 Cormier SA, Lomnicki S, Backes W, Dellinger

laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Lett. 239(1), 129135 (2006).
32 Brown C, Bushell G, Whitehouse M, Agrawal

B. Origin and health impacts of emissions of toxic by-products and fine particles from combustion and thermal treatment of hazardous wastes and materials. Environ. Health Perspect. 114(6), 810817 (2006).
19 Bonde JP, Ramlau-Hansen CH, Olsen

D, Tupe S, Paknikar K. Nanogoldpharmaceutics: (i) the use of colloidal gold to treat experimentally-induced arthritis in rat models; (ii) characterization of the gold in Swarna bhasma , a microparticulate used in traditional Indian medicine. Gold Bull. 40(3), 245250 (2007).
33 Tsai C, Shiau A, Chen S, Chen Y, Cheng P,

J.Trends in sperm counts: the saga continues. Epidemiology 22(5), 617619 (2011).
20 Shi L, Yang R, Yue W etal. Effect of elemental

nano-selenium quality, glutathione peroxidase activity, and testis ultrastructure in male Boer goats. Anim. Reprod. Sci. 118(24), 248254 (2010).
21 Dym M, Fawcett DW. The bloodtestis

Chang M. Amelioration of collagen-induced arthritis in rats by nanogold. Arthritis Rheum. 56(2), 544554 (2007).
34 Balasubramanian S, Jittiwat J, Manikandan J,

barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3(3), 308326 (1970).
22 Shi L, Xun W, Yue W etal. Effect of sodium

Ong C, Yu L, Ong W. Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration in rat. Biomaterials 31(8), 20342042 (2010).
35 De Jong WH, Hagens WI, Krystek P, Burger

selenite, Se-yeast and nano-elemental selenium on growth performance, Se concentration and antioxidant status in growing male goats. Small Ruminant Res. 96(1), 4952 (2011).
23 Park EJ, Bae E, Yi J etal. Repeated-dose

MC, Sips AJ, Geertsma RE. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 29(12), 19121919 (2008).
36 Makhluf SBD, Qasem R, Rubinstein S,

10 Johnston HJ, Hutchison GR, Christensen FM,

toxicity and inflammatory responses in mice by oral administration of silver nanoparticles. Environ. Toxicol. Pharmacol. 30(2), 162168 (2010).
n

Gedanken A, Breitbart H. Loading magnetic nanoparticles into sperm cells does not affect their functionality. Langmuir 22(23), 94809482 (2006).
37 Kim JS, Yoon TJ, Yu KN etal. Toxicity and

11 Panyala N, Pena-Mendez E, Havel J.Silver or

The elevator hypothesis was generated based on data from this study. Toxicology. Hayes AW (Ed.). CRC Press/ Taylor & Francis, PA, USA (2008).

tissue distribution of magnetic nanoparticles in mice. Toxicol. Sci. 89(1), 338347 (2006).
38 Kwon JT, Hwang SK, Jin H etal. Body

silver nanoparticles: a hazardous threat to the environment and human health. J.Appl. Biomed. 6, 117129 (2008).
12 Schrand AM, Rahman MF, Hussain SM,

24 Hayes AW. Principles and Methods of

Schlager JJ, Smith DA, Syed AF. Metal-based nanoparticles and their toxicity assessment. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2(5), 544568 (2010).
13 Hussain SM, Braydich-Stolle LK, Schrand AM

25 Foster L, Sumar S. Selenium in health and

distribution of inhaled fluorescent magnetic nanoparticles in the mice. J.Occup. Health 50(1), 16 (2008).
39 Cho YW, Park SA, Han TH etal. Invivo

disease: a review. Crit. Rev. Food Sci. Nutr. 37(3), 211228 (1997).
26 Ip C. Lessons from basic research in selenium

etal. Toxicity evaluation for safe use of nanomaterials: recent achievements and technical challenges. Adv. Mater. 21(16), 15491559 (2009).
14 Bonde JP. Male reproductive organs are at risk

and cancer prevention. J.Nutr. 128(11), 18451854 (1998).

27 Spallholz J.On the nature of selenium

tumor targeting and radionuclide imaging with self-assembled nanoparticles: mechanisms, key factors, and their implications. Biomaterials 28(6), 12361247 (2007).
40 Martin L, Wilson CG, Koosha F etal. The

toxicity and carcinostatic activity. Free Radic. Biol. Med. 17(1), 4564 (1994).
28 Gao X, Zhang J, Zhang L. Hollow sphere

release of model macromolecules may be controlled by the hydrophobicity of palmitoyl glycol chitosan hydrogels. J.Control. Release 80(13), 87100 (2002).
41 Hyung PJ, Kwon S, Lee M etal. Self-assembled

from environmental hazards. Asian J.Androl. 12(2), 152156 (2010).

15 Auger J, Kunstmann JM, Czyglik F, Jouannet

selenium nanoparticles: their invitro anti hydroxyl radical effect. Adv. Mater. 14(4), 290293 (2002).
29 Jia X, Li N, Chen J.A subchronic toxicity

P. Decline in semen quality among fertile men in Paris during the past 20years. N. Engl. J.Med. 332(5), 281285 (1995).

study of elemental Nano-Se in SpragueDawley rats. Life Sci. 76(17), 19892003 (2005).

nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: invivo biodistribution and anti-tumor activity. Biomaterials 27(1), 119126 (2006).

594

Nanomedicine (2012) 7 (4)

future science group

How nanoparticles affect spermatogenesis

Review

42 Kim JH, Kim YS, Park K etal. Anti-tumor

54 Kim YS, Song MY, Park JD etal. Subchronic

efcacy of cisplatin-loaded glycol chitosan nanoparticles in tumor-bearing mice. J.Control. Release 127(1), 4149 (2008).
43 Choi M, Cho M, Han B etal. Chitosan

oral toxicity of silver nanoparticles. Part. Fibre Toxicol. 7, 20 (2010).

55 Wolf R, Matz H, Orion E, Lipozenci J.
nn

testosterone biosynthesis and metabolism via growth hormone. Toxicol. Lett. 191(23), 103108 (2009). This paper can be regarded as a research example of an in vivo study aimed at exploring nanoparticle toxicity on spermatogenesis. The effects of nanoparticles on mouse testis Leydig cells invitro. Toxicol. InVitro 22(8), 18251831 (2008).
nn

nanoparticles show rapid extrapulmonary tissue distribution and excretion with mild pulmonary inflammation to mice. Toxicol. Lett. 199(2), 144152 (2010).
44 Lacerda L, Bianco A, Prato M, Kostarelos K.

Sunscreensthe ultimate cosmetic. Acta Dermatovenerol. Croat. 11(3), 158162 (2003).

56 Sayes CM, Wahi R, Kurian PA etal.

Carbon nanotubes as nanomedicines: from toxicology to pharmacology. Adv. Drug Deliv. Rev. 58(14), 14601470 (2006).
45 Liu Z, Cai W, He L etal. Invivo

Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol. Sci. 92(1), 174185 (2006).
57 Li H, Luo W, Tao Y etal. Effects of

67 Komatsu T, Tabata M, Kubo-Irie M etal.

biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2(1), 4752 (2007).
46 Chen J, Chen S, Zhao X, Kuznetsova LV, Wong

SS, Ojima I. Functionalized single-walled carbon nanotubes as rationally designed vehicles for tumor-targeted drug delivery. J.Am. Chem. Soc. 130(49), 1677816785 (2008).
47 Usui Y, Aoki K, Narita N etal. Carbon

nanoscale quantum dots in male Chinese loaches (Misgurnus anguillicaudatus ): estrogenic interference action, toxicokinetics and oxidative stress. Sci. China Ser. B Chem. 52(10), 16831690 (2008).
58 Wang J, Li N, Zheng L etal. P38-Nrf-2

This paper can be regarded as research example in vitro study which is aimed at supplementing in vivo studies and exploring molecular mechanisms more efficiently. Rojanathanes R. Effect of gold nanoparticles on spermatozoa: the rst world report. Fertil. Steril. 91(1), e7e8 (2009).

68 Wiwanitkit V, Sereemaspun A,

signaling pathway of oxidative stress in mice caused by nanoparticulate TiO2. Biol. Trace Elem. Res. 140(2), 186197 (2011).
59 Gurr JR, Wang AS, Chen CH, Jan KY.

69 Cheng CY, Mruk DD. Cell junction

nanotubes with high bone-tissue compatibility and bone-formation acceleration effects. Small 4(2), 240246 (2008).
48 Yoshida S, Hiyoshi K, Ichinose T etal. Effect

of nanoparticles on the male reproductive system of mice. Int. J.Androl. 32(4), 337342 (2009).
49 Bai Y, Zhang Y, Zhang J etal. Repeated

Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213(12), 6673 (2005).
60 Takeda K, Suzuki K, Ishihara A etal.

dynamics in the testis: sertoli-germ cell interactions and male contraceptive development. Physiol. Rev. 82(4), 825874 (2002).
70 Furuse M, Sasaki H, Fujimoto K, Tsukita S.

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J.Cell Biol. 143(2), 391401 (1998).
71 Hellani A, Ji J, Mauduit C, Deschildre C,

administrations of carbon nanotubes in male mice cause reversible testis damage without affecting fertility. Nat. Nanotechnol. 5(9), 683689 (2010).
nn

Nanoparticles transferred from pregnant mice to their offspring can damage the genital and cranial nerve systems. J.Health 55, 95102 (2009).
61 Bal R, Trk G, Tuzcu M etal. Protective

This paper can be regarded as a research example in vivo study which is aimed at exploring nanoparticle toxicity on spermatogenesis. Pharmacology of Silver. Williams & Wilkins Company, MD, USA (1939).

effects of nanostructures of hydrated C 60 fullerene on reproductive function in streptozotocin-diabetic male rats. Toxicology 282(3), 6981 (2011).
62 Igeolet E, Corbisier P, Houbion A etal.

Tabone E, Benahmed M. Developmental and hormonal regulation of the expression of oligodendrocyte-specific protein/claudin 11 in mouse testis. Endocrinology 141(8), 30123019 (2000).
72 Fijak M, Bhushan S, Meinhardt A.

Immunoprivileged sites: the testis. Methods Mol. Biol. 677, 459470 (2011).
73 Krawetz SA, De Rooij DG, Hedger MP.

50 Hill W, Pillsbury D. Argyria: The

51 Hussain SM, Javorina AK, Schrand AM,

Duhart HM, Ali SF, Schlager JJ.The interaction of manganese nanoparticles with pc-12 cells induces dopamine depletion. Toxicol. Sci. 92(2), 456463 (2006).
52 Hussain SM, Hess KL, Gearhart JM, Geiss

Glutathione peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived free radicals. Mech. Ageing Dev. 51(3), 283297 (1990).
63 Lin BC, Xi ZG, Zhang YG. Oxidative

Molecular aspects of male fertility. International workshop on molecular andrology. EMBO Rep. 10(10), 10871092 (2009).
74 Fijak M, Meinhardt A. The testis in

damage on testicles of male rats induced by micro-nano-scale SiO2. J.Environ. Health 24(8), 574576 (2007).
64 Shrilatha B, Muralidhara. Early oxidative

immune privilege. Immunol. Rev. 213, 6681(2006).

75 Meng J, Holdcraft RW, Shima JE, Griswold

KT, Schlager JJ.Invitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol. Invitro 19(7), 975983 (2005).
53 Braydich-Stolle L, Hussain S, Schlager JJ,

Hofmann MC. Invitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol. Sci. 88(2), 412419 (2005).
nn

stress in testis and epididymal sperm in streptozotocin-induced diabetic mice: its progression and genotoxic consequences. Reprod. Toxicol. 23(4), 578587 (2007).
65 Fan YO, Zhang YH, Zhang XP, Liu B.

MD, Braun RE. Androgens regulate the permeability of the bloodtestis barrier. Proc. Natl Acad. Sci. USA 102(46), 1669616700 (2005).
76 Warren DW, Pasupuleti V, Lu Y, Platler BW,

This paper can be regarded as research example in vitro study which is aimed at supplementing in vivo studies and exploring molecular mechanisms more efficiently.

Comparative study of nanosized and microsized silicon dioxide on spermatogenesis function of male rats. Wei Sheng Yan Jiu 35(5), 549553 (2006).
66 Ramdhan DH, Ito Y, Yanagiba Y.

Horton R. Tumor necrosis factor and interleukin-1 stimulate testosterone secretion in adult male rat Leydig cells invitro. J.Androl. 11(4), 353360 (1990).
77 Delfino FJ, Boustead JN, Fix C, Walker WH.

Nanoparticle-rich diesel exhaust may disrupt

NF- B and TNF- stimulate androgen receptor expression in sertoli cells. Mol. Cell. Endocrinol. 201(12), 112 (2003).

future science group

www.futuremedicine.com

595

Review

Lan & Yang

78 Zhang Y, Ling F, Zhu Q etal. New

81 Fernandes C, Soni U, Patravale V. Nano-

84 Misra A, Ganesh S, Shahiwala A, Shah SP.

development of study on the toxicity of nano materials. Nanoscience 11, 137141 (2006).
79 Podio V, Zara GP, Carazzone M, Cavalli R,

interventions for neurodegenerative disorders. Pharmacol. Res. 62(2), 166178 (2010).

82 Nag S. Morphology and properties of brain

Drug delivery to the central nervous system: a review. J.Pharm. Sci. 6(2), 252273 (2003).

Gasco MR. Biodistribution of stealth and non-stealth solid lipid nanospheres after intravenous administration to rats. J.Pharm. Pharmacol. 52(9), 10571063 (2002).
80 Hasirci N. Micro and nano systems in

endothelial cells. Methods Mol. Biol. 686, 347 (2011).

83 Sharom FJ.ABC multidrug transporters:

Website
101 University of Adelaide. Department of

biomedicine and drug delivery. Nanoparticles Nanosystems Biomed. Appl. 126 (2007).

structure, function and role in chemoresistance. Pharmacogenomics 9(1), 105127 (2008).

Chemistry. Inductively-Coupled Plasma (ICP) Excitation Source. www.chemistry.adelaide.edu.au/external/ soc-rel/content/icp.htm

596

Nanomedicine (2012) 7 (4)

future science group

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.