**6. Pharmacokinetics of cadmium-containing quantum dots**

**5. Cadmium-containing quantum dots as a platform for nanoparticle drug**

Recently, there has been an explosion in the development of nanoparticle-based drug delivery vehicles composed of lipids, polymers, carbon materials, and even hybrid combinations of those materials tailored not only for the dramatic improvement of the pharmacological properties of existing drugs, but also for enabling the delivery of new classes of potent anticancer drugs for gene therapy and immunotherapy [67, 68]. With QDs, a combination of unique physical, chemical, and optical properties facilitates in-depth study of nanocarrier interactions with biological systems through real-time monitoring of QDs biodistribution, intracellular uptake, drug release, and long-term nanocarrier fate. At the same time, compact size and compatibility with a variety of surface modification strategies enables substitution of virtually any QD core with a QD within single-nanoparticle drug delivery vehicles, or incorporation of QD tags within larger multicomponent vehicles. The combination of superior brightness and resistance to photo-degradation represents another set of QD properties that highly useful for

The great interest in engineering NP-based drug delivery vehicles is driven by the powerful capability of nanocarriers to completely redefine the pharmacokinetic properties of virtually any drug, ranging from small-molecule therapeutics to large proteins and DNA plasmids. Encapsulation of the drug within the nanoparticles keeps it shielded from the biological environment until the moment of carrier degradation and drug release, thus minimizing non-

A few reports have appeared recently regarding this ambitious goal. It has previously conjugated captopril, an antihypertensive drug, to the QD surface and studied its pharmaco‐ dynamics and pharmacokinetics in stroke-prone spontaneously hypertensive rats [70]. The results show that the administered QD-captopril conjugates are capable of decreasing rat blood pressure to the same extent as the captopril alone in the first 30 min, but the therapeutic effect of QD-captopril disappears after 60 min. It is unclear whether the therapeutic effect results from the QD-captopril conjugates or captopril molecules detached from the QD surface. Another piece of interesting work was previously reported, wherein a targeting functionality was added to QDs by linking them with RNA aptamers (A10) that specifically bind to prostate specific membrane antigen (PSMA) [71]. Doxorubicin, a DNA-interacting drug widely used in chemotherapy, was immobilized onto QDs by intercalation within the A10 RNA aptamer [72]. Another study reported the ability of nanoconjugates of CdSe/CdS/ZnS and doxorubicin (Dox) to target alveolar macrophages, cells that play a critical role in the pathogenesis of inflammatory lung injuries. The results demonstrated that nanoparticle platforms can provide targeted macrophage-selective therapy for the treatment of pulmonary disease [73]. This was

previously reported regarding siRNA delivery using QDs as delivery vehicles [74].

QDs already play an important role in fundamental biology and *in vitro* disease diagnostics and prognostics. Their unique structural and surface properties, such as their tunable and uniform size, flexible drug-linking and doping mechanisms, large surface-to-volume ratio and wide spectrum of surface reactive groups have enabled a new avenue of research: targeted

specific and potentially adverse interactions en route to the target [69].

**delivery vehicle design**

478 Application of Nanotechnology in Drug Delivery

long-term nanocarrier tracking.

Nanoparticle-based drug delivery is on its way to overcoming the fundamental limitations of simple free drug formulations, providing means to change their pharmacological properties and also understand their biological fate in great detail. Among many contrast agents for studying nanoparticle-based drug delivery vehicles, QDs are particularly suitable. Their unique amalgamation of useful features, such as small size, versatile surface chemistry, and exquisite optical properties make them an ideal platform for the comprehensive characteriza‐ tion of nanoparticle-based drug delivery vehicle behavior across single-cell to whole organism levels. In this new field, QDs have already made substantial contributions, enabling dynamic monitoring of nanocarrier cell uptake, intracellular distribution, circulation half-times, and biodistribution. Early on, the design of QDs drug delivery vehicles was governed by the intrinsically poor pharmacokinetic (PK) properties of conventional drugs. Low drug solubility, rapid metabolism and clearance and, most importantly, a lack of selectivity, regularly lead to therapeutic failure by causing severe systemic toxicity in healthy tissues, thus prohibiting the dose escalation necessary to eliminate tumor cells. Incorporating these drugs into nanocarriers offers an exciting opportunity to redefine the PK properties, improving therapeutic efficacy and reducing side effects.

When we utilize a nanopharmaceutical, it is important to realize that, in contrast to delivering a drug that is an organic molecule, we are delivering something of a discrete entity in a nanoparticle-comprised of atomic scale parts. Due to the quantum effects and electronic interactions that predominate at the nanoscale, we need to alter the way in which we think about pharmacological parameters in order to adapt to nanoscience. Nanopharmacology is further complicated by the need to establish the behavior of nanoparticles such as QDs within the traditional pharmacological parameters of absorption, distribution, metabolism and excretion (ADME). Nanoconstructs, in many cases, have limited metabolism and excretion and persist in biological systems; this becomes particularly important when toxic atoms such as cadmium are involved. The pharmacological parameters of the behavior of cadmiumcontaining quantum dots in biological systems is currently still under investigation. Dosing parameters, absorption, distribution, metabolism and excretion require considerable further study, since we have little information on these parameters at this point.

The first factor we need to establish before we study the pharmacokinetics parameter is the dose. Adequate estimates of QDs exposure during treatments such as cancer therapy must be in place so that both pharmacological and toxicological studies can be conducted within a physiologically relevant dose range. Importantly, how does cadmium content relate to dose? To date, these parameters have not been rigorously addressed. Dose metrics of QDs have been reported in terms of mg/kg (or mg/ml for *in vitro* studies) or on a molar basis, and this will no doubt need refinement. Given that a QD, or any other nanoparticle, is an engineered entity or "mini-reactor", dose via mass or molar number may be an inappropriate descriptor. The length and extent of QDs tissue retention is an important parameter for dosing considerations, since repeated doses may induce systemic accumulation, contributing to potential toxicity. Further‐ more, persistence of QDs in tissues demands long-term studies to accurately assess risk. Therefore, the dose is another parameter that requires consideration.

that specific QDs influence corneal stromal cell viability up to a significant magnitude of 50% in a relatively low concentration (5-20 nM) and under a short exposure period (24-48 h) [82]. QDs can also be retained in the cornea up to 26 days in an *in vivo* mouse model. Therefore, since corneal stromal cells are crucial for the maintenance of the health and transparency of the cornea, potential QDs cytotoxicity due to bioaccumulation is a major concern given the

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Absorption via tissues and biological fluids is generally the first hurdle to be met and this is, of course, dependent on route of delivery. Currently, the most important route of delivery for QDs appears to be systemic distribution through parenteral delivery [83]. Nanoparticles are considered to absorb more effectively into the respiratory, skin, and gastrointestinal systems than micron-sized particles because of their unique physicochemical properties, such as their size and surface modifications [84]. For example, instillation of 5 nm QDs shows faster translocation to other organs than in the case of 27 nm QDs [85]. In addition, a recent study showed that nanoparticles were only translocated into the circulation system when adminis‐ tered into the lung; their absorption rate varied, depending on their surface properties [86]. When different sizes of polystyrene QDs were administered orally to rats, the absorption of 50–100 nm polystyrene QDs was about 250-fold higher than the absorption of larger micro‐ particles (500 nm, 1, and 10 μm) [87]. Oral administration of different sizes of colloidal gold particles to mice showed size-dependent absorption [88]. Regarding surface modification, positively charged particles show better absorption than neutral or negatively charged particles [89]. However, in a recent study using gold nanoparticles, negatively charged particles showed higher absorption rates than positively charged particles [90]. Therefore, no general rule can be made about absorption from these results and further studies on the impact of surface modification on gastrointestinal absorption are required. Studying the kinetics of

Regarding distribution, one of the first elements that parenteral delivered QD will encounter is the environment of the blood. Here, we have little to no information about blood/QD interactions. Plasma half-life is no doubt related to surface coating and addition of biological targeting ligands, if any. For example, PEG coating was reported to increase plasma half-life [91]. In the case of oxide nanoparticles, many are coated with endogenous plasma proteins immediately upon entry into the circulatory system and this appears to enhance tissue delivery. However, the interactions between QDs and plasma proteins are unknown. Immune responses may also be triggered at this level, as reviewed by [92], but the precise interactions of QDs at this level have not been examined. Recently, some studies have investigated the *in vivo* biodistribution of QDs [93-95]. All of the reports in the literature unanimously conclude that QDs show a preference for deposition in organs and tissues and that they do not remain circulating in the bloodstream [96]. Real-time imaging of live animals treated with the hyaluronic acid (HA)-conjugated QDs showed that the luminescence of NIR QDs could be detected for up to 2 months [97]. Others reported that QDs coated using an amphiphilic

poly(acrylic acid) polymer remain fluorescent after at least four months *in vivo* [98].

Organ-selective biodistribution and elimination routes of synthesized QDs coated with 3 mercaptopropionic acid (QD MPA) and commercially available Qtracker 705 nontargeted

potential threat to corneal health.

nanoparticles is an important issue in nanotechnology.

The second factor to establish is the route of administration. This, along with the risks for exposure and possible toxicity should be addressed cautiously to ensure experimental and clinical safety. Moreover, an understanding of possible interactions of QDs within human tissues may shed light on the prevention of health risks in the laboratory and daily exposure to QDs. To date, research has established that various nanoparticles can enter an organism through skin, inhalation, oral delivery, and parenteral administration. Intravenous injection is a major route for drug administration. It has been reported that intravenously injected QDs may accumulate in unintended tissues, which implies that the potential toxicity might result from a failure to clear them from the body [76]. The possibility of tissue uptake of QDs via ingestion following exposure has also been proposed in animal and cell culture models. Skin is another important portal of entry for nanoparticles as potential route of systemic drug administration.

The abilities of nanoagents to penetrate the skin barrier are debatable. Some studies indicate that NP penetrate skin barrier under skin lesions, UV sunburn or ultrasound [77]. The potential beneficial and/or side effects of intradermally accumulated NP primarily depend on their localization in the tissues. It is also important to elucidate further migration pathways of particles, as this would determine their pharmacokinetic properties and systemic distribution in the organism. Topically applied QDs were reported to accumulate in the dermis and muscles [78]; however, a more detailed localization of particles has not been described. Other studies reported that the majority of the intradermally injected QDs remained at the injection site and a minority of QDs migrated into lymph nodes, highlighting QDs advantages for lym-phatic imaging [79, 80]. However, high retention of QDs at the injection site raises concerns about long-term physiological effects. There is a lack of in-depth analyses of QDs localization and migration pathways in the dermis. It was recently reported that the subcutaneous injection of CdSe/ZnS coated with mPEG-5000 polymer into the CDF1 of mice showed that basement membrane and dense connective tissue fibers limited the diffusion of QDs in the dermis [81]. Negligible QDs penetration into the epidermis, hair follicles, sebaceous and sweat glands, nerves and blood vessels was also observed. However, low permeation of QDs through the tissues could result in slow clearance and raises the risks of potential immune, inflammatory and cytotoxic responses.

The cornea is another common route of drug administration and also an important route of nanoparticle exposure in occupational scenarios and daily life. However, the possibility of QDs penetration into the cornea has not been discussed before. It has been previously demonstrated that specific QDs influence corneal stromal cell viability up to a significant magnitude of 50% in a relatively low concentration (5-20 nM) and under a short exposure period (24-48 h) [82]. QDs can also be retained in the cornea up to 26 days in an *in vivo* mouse model. Therefore, since corneal stromal cells are crucial for the maintenance of the health and transparency of the cornea, potential QDs cytotoxicity due to bioaccumulation is a major concern given the potential threat to corneal health.

physiologically relevant dose range. Importantly, how does cadmium content relate to dose? To date, these parameters have not been rigorously addressed. Dose metrics of QDs have been reported in terms of mg/kg (or mg/ml for *in vitro* studies) or on a molar basis, and this will no doubt need refinement. Given that a QD, or any other nanoparticle, is an engineered entity or "mini-reactor", dose via mass or molar number may be an inappropriate descriptor. The length and extent of QDs tissue retention is an important parameter for dosing considerations, since repeated doses may induce systemic accumulation, contributing to potential toxicity. Further‐ more, persistence of QDs in tissues demands long-term studies to accurately assess risk.

The second factor to establish is the route of administration. This, along with the risks for exposure and possible toxicity should be addressed cautiously to ensure experimental and clinical safety. Moreover, an understanding of possible interactions of QDs within human tissues may shed light on the prevention of health risks in the laboratory and daily exposure to QDs. To date, research has established that various nanoparticles can enter an organism through skin, inhalation, oral delivery, and parenteral administration. Intravenous injection is a major route for drug administration. It has been reported that intravenously injected QDs may accumulate in unintended tissues, which implies that the potential toxicity might result from a failure to clear them from the body [76]. The possibility of tissue uptake of QDs via ingestion following exposure has also been proposed in animal and cell culture models. Skin is another important portal of entry for nanoparticles as potential route of systemic drug

The abilities of nanoagents to penetrate the skin barrier are debatable. Some studies indicate that NP penetrate skin barrier under skin lesions, UV sunburn or ultrasound [77]. The potential beneficial and/or side effects of intradermally accumulated NP primarily depend on their localization in the tissues. It is also important to elucidate further migration pathways of particles, as this would determine their pharmacokinetic properties and systemic distribution in the organism. Topically applied QDs were reported to accumulate in the dermis and muscles [78]; however, a more detailed localization of particles has not been described. Other studies reported that the majority of the intradermally injected QDs remained at the injection site and a minority of QDs migrated into lymph nodes, highlighting QDs advantages for lym-phatic imaging [79, 80]. However, high retention of QDs at the injection site raises concerns about long-term physiological effects. There is a lack of in-depth analyses of QDs localization and migration pathways in the dermis. It was recently reported that the subcutaneous injection of CdSe/ZnS coated with mPEG-5000 polymer into the CDF1 of mice showed that basement membrane and dense connective tissue fibers limited the diffusion of QDs in the dermis [81]. Negligible QDs penetration into the epidermis, hair follicles, sebaceous and sweat glands, nerves and blood vessels was also observed. However, low permeation of QDs through the tissues could result in slow clearance and raises the risks of potential immune, inflammatory

The cornea is another common route of drug administration and also an important route of nanoparticle exposure in occupational scenarios and daily life. However, the possibility of QDs penetration into the cornea has not been discussed before. It has been previously demonstrated

Therefore, the dose is another parameter that requires consideration.

administration.

480 Application of Nanotechnology in Drug Delivery

and cytotoxic responses.

Absorption via tissues and biological fluids is generally the first hurdle to be met and this is, of course, dependent on route of delivery. Currently, the most important route of delivery for QDs appears to be systemic distribution through parenteral delivery [83]. Nanoparticles are considered to absorb more effectively into the respiratory, skin, and gastrointestinal systems than micron-sized particles because of their unique physicochemical properties, such as their size and surface modifications [84]. For example, instillation of 5 nm QDs shows faster translocation to other organs than in the case of 27 nm QDs [85]. In addition, a recent study showed that nanoparticles were only translocated into the circulation system when adminis‐ tered into the lung; their absorption rate varied, depending on their surface properties [86]. When different sizes of polystyrene QDs were administered orally to rats, the absorption of 50–100 nm polystyrene QDs was about 250-fold higher than the absorption of larger micro‐ particles (500 nm, 1, and 10 μm) [87]. Oral administration of different sizes of colloidal gold particles to mice showed size-dependent absorption [88]. Regarding surface modification, positively charged particles show better absorption than neutral or negatively charged particles [89]. However, in a recent study using gold nanoparticles, negatively charged particles showed higher absorption rates than positively charged particles [90]. Therefore, no general rule can be made about absorption from these results and further studies on the impact of surface modification on gastrointestinal absorption are required. Studying the kinetics of nanoparticles is an important issue in nanotechnology.

Regarding distribution, one of the first elements that parenteral delivered QD will encounter is the environment of the blood. Here, we have little to no information about blood/QD interactions. Plasma half-life is no doubt related to surface coating and addition of biological targeting ligands, if any. For example, PEG coating was reported to increase plasma half-life [91]. In the case of oxide nanoparticles, many are coated with endogenous plasma proteins immediately upon entry into the circulatory system and this appears to enhance tissue delivery. However, the interactions between QDs and plasma proteins are unknown. Immune responses may also be triggered at this level, as reviewed by [92], but the precise interactions of QDs at this level have not been examined. Recently, some studies have investigated the *in vivo* biodistribution of QDs [93-95]. All of the reports in the literature unanimously conclude that QDs show a preference for deposition in organs and tissues and that they do not remain circulating in the bloodstream [96]. Real-time imaging of live animals treated with the hyaluronic acid (HA)-conjugated QDs showed that the luminescence of NIR QDs could be detected for up to 2 months [97]. Others reported that QDs coated using an amphiphilic poly(acrylic acid) polymer remain fluorescent after at least four months *in vivo* [98].

Organ-selective biodistribution and elimination routes of synthesized QDs coated with 3 mercaptopropionic acid (QD MPA) and commercially available Qtracker 705 nontargeted quantum dots with poly(ethylene glycol) coating (QD PEG) were recently compared after intravenous injection to mice. QDs were deposited mainly in liver, spleen, kidney and lymph nodes [99]. Another study observed that, in the murine model, quantum dots (CdSe 100 pmol/ kg) administered by intravenous injection mainly accumulated in the red pulps of the spleen, portal areas of the liver, and adrenal glands of the kidney within 1 h. It is believed that quantum dots do not degrade, as evidenced by the intensity of the fluorescence in the fluorescence spectrum image and comparable intensity profile of Cd [100]. One consistent theme in the existing literature is that QDs are eventually taken up by the reticuloendothelial system, including the liver, spleen, and lymphatic system [101]. On the other hand, it has been found that aqQDs are initially accumulated in liver after short-time (0.5- 4h) post-injection, and then are increasingly absorbed by kidney during long-time (15 – 80 days) blood circulation. Moreover, size-dependent biodistribution is obviously observed: aqQDs with larger sizes are more quickly accumulated in the spleen [102]. We have recently found that after i.p. injection, the CdS/Maltodextrin quantum dots mainly accumulated in the liver, kidney, spleen, thymus, intestine, lung and brain (Figure 3). The pharmacokinetics studies showed the presence of CdS/ Maltodextrin in kidney 30 min after administration; at 18 h, the presence of quantum dots in kidney was more abundant. However, the amount of CdS/Maltodextrin quantum dots in liver was lower than in kidney, and the maximal presence was at 3 h. Our results suggest that the transition of CdS/Maltodextrin quantum dots via the liver is very fast, and the main route of elimination is renal (Figure 4).

**Figure 4.** Biokinetics of excretion of CdS/maltodextrin quantum dots after a single i.p. administration of 10 μg in kid‐

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The blood–brain barrier (BBB), is a critical interface and acts as a physical and metabolic barrier between the CNS and the peripheral circulation that serves to regulate and protect the microenvironment of the brain. The primary function of the normal BBB is to establish and maintain homeostasis in the CNS [103]. Under normal physiological conditions, BBB prevents transport of bacteria, large molecules, and most small molecules into the brain. To be BBB permeable, molecules need to be lipid soluble and less than 400 Da in size. There are more than 7000 drugs in the comprehensive medicinal chemistry database, and only 5% of these drugs can be used to treat the CNS diseases. In a related study, 12% of all drugs are shown to be active in the CNS, but only 1% of all drugs are active in the brain for diseases other than affective disorders [104]. The importance of developing new approaches to brain drug development is illustrated by considering the limitations of the existing brain drug delivery strategies. Therefore, there is great interest in nanomedicine to develop optimized traceable drug-loaded nanoparticle formulations that can be safely used to facilitate drugs across the BBB for treating brain diseases and, at the same time, visualizing the distribution profile of the nanoparticles in the brain. The use of QDs bioconjugates as efficient targeted probes for transmigration across the BBB has been previously demonstrated [105]. Since the transferrin receptor protein is highly localized on the endothelial surface of the brain, transferrin (Tf) was selected to trigger receptor-mediated transport across the BBB. It was discovered that the migration rate of Tfconjugated QDs crossing the *in vitro* BBB is both concentration-and time-dependent [106]. It was recently demonstrated that the QDs-Tf-Saquinavir nanoformulation increases the drug solubility, enhancing systemic bioavailability, while the excellent optical properties of QDs

ney and liver.

**Figure 3.** Biodistribution of CdS/Maltodextrin in Wistar rats. Tissue distribution of CdS/maltodextrin quantum dots af‐ ter i.p. administration of 10 μg during 8 days. Quantum dots were observed in all analyzed tissues, including brain. The maximal observed presence of quantum dots was in lung and kidney.

quantum dots with poly(ethylene glycol) coating (QD PEG) were recently compared after intravenous injection to mice. QDs were deposited mainly in liver, spleen, kidney and lymph nodes [99]. Another study observed that, in the murine model, quantum dots (CdSe 100 pmol/ kg) administered by intravenous injection mainly accumulated in the red pulps of the spleen, portal areas of the liver, and adrenal glands of the kidney within 1 h. It is believed that quantum dots do not degrade, as evidenced by the intensity of the fluorescence in the fluorescence spectrum image and comparable intensity profile of Cd [100]. One consistent theme in the existing literature is that QDs are eventually taken up by the reticuloendothelial system, including the liver, spleen, and lymphatic system [101]. On the other hand, it has been found that aqQDs are initially accumulated in liver after short-time (0.5- 4h) post-injection, and then are increasingly absorbed by kidney during long-time (15 – 80 days) blood circulation. Moreover, size-dependent biodistribution is obviously observed: aqQDs with larger sizes are more quickly accumulated in the spleen [102]. We have recently found that after i.p. injection, the CdS/Maltodextrin quantum dots mainly accumulated in the liver, kidney, spleen, thymus, intestine, lung and brain (Figure 3). The pharmacokinetics studies showed the presence of CdS/ Maltodextrin in kidney 30 min after administration; at 18 h, the presence of quantum dots in kidney was more abundant. However, the amount of CdS/Maltodextrin quantum dots in liver was lower than in kidney, and the maximal presence was at 3 h. Our results suggest that the transition of CdS/Maltodextrin quantum dots via the liver is very fast, and the main route of

**Figure 3.** Biodistribution of CdS/Maltodextrin in Wistar rats. Tissue distribution of CdS/maltodextrin quantum dots af‐ ter i.p. administration of 10 μg during 8 days. Quantum dots were observed in all analyzed tissues, including brain. The

maximal observed presence of quantum dots was in lung and kidney.

elimination is renal (Figure 4).

482 Application of Nanotechnology in Drug Delivery

**Figure 4.** Biokinetics of excretion of CdS/maltodextrin quantum dots after a single i.p. administration of 10 μg in kid‐ ney and liver.

The blood–brain barrier (BBB), is a critical interface and acts as a physical and metabolic barrier between the CNS and the peripheral circulation that serves to regulate and protect the microenvironment of the brain. The primary function of the normal BBB is to establish and maintain homeostasis in the CNS [103]. Under normal physiological conditions, BBB prevents transport of bacteria, large molecules, and most small molecules into the brain. To be BBB permeable, molecules need to be lipid soluble and less than 400 Da in size. There are more than 7000 drugs in the comprehensive medicinal chemistry database, and only 5% of these drugs can be used to treat the CNS diseases. In a related study, 12% of all drugs are shown to be active in the CNS, but only 1% of all drugs are active in the brain for diseases other than affective disorders [104]. The importance of developing new approaches to brain drug development is illustrated by considering the limitations of the existing brain drug delivery strategies. Therefore, there is great interest in nanomedicine to develop optimized traceable drug-loaded nanoparticle formulations that can be safely used to facilitate drugs across the BBB for treating brain diseases and, at the same time, visualizing the distribution profile of the nanoparticles in the brain. The use of QDs bioconjugates as efficient targeted probes for transmigration across the BBB has been previously demonstrated [105]. Since the transferrin receptor protein is highly localized on the endothelial surface of the brain, transferrin (Tf) was selected to trigger receptor-mediated transport across the BBB. It was discovered that the migration rate of Tfconjugated QDs crossing the *in vitro* BBB is both concentration-and time-dependent [106]. It was recently demonstrated that the QDs-Tf-Saquinavir nanoformulation increases the drug solubility, enhancing systemic bioavailability, while the excellent optical properties of QDs also visualize the distribution and accumulation of nanoplexes in brain [107]. Such primary results offer a basis for the development of novel QDs with targeting molecules and drugs, which could enhance drug delivery efficacy across the BBB and facilitate the uptake of the QDdrugs in the brain. Despite the encouraging results on the use of QDs for drug delivery across the BBB, there is still serious concern regarding QDs toxicity *in vivo,* which mainly originates from the intrinsic, potentially toxic nature of the semiconductor materials themselves.

On the other hand, the placental barrier protects the embryo from various chemical agents and other foreign substances in the body. However, the passage of xenobiotic molecules through the placental barrier is not completely prevented, and drugs may affect fetal cell proliferation, embryonic growth, and organ formation [108]. Knowledge regarding embryotoxicity is of great importance because it is a necessary part of the toxicological profile that must be established for any new biologically active substance relevant to human safety. There are many studies in this field [109-111], but the detailed effects of NPs still pose many questions. A decrease in embryonic weight after QDs injection on the sixth day of embryogenesis (CdSe/ZnS QDs, 9.6%; CdT QDs, 6.2%) has been reported, and no embryotoxic or teratogenic effects were observed at any stage of embryogenesis [112]. Others have shown that NPs (nSP and TiO2s 70 nm and 35 nm in diameter, respectively) can cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring [113]. They showed that the NPs were found in the placenta, fetal liver, and fetal brain. Other authors argue that some NPs (CdSe and CdTe/CdS) in different sizes, at different dosages, and with different outer capping materials can increase the rate of early-stage blastocyst death in mice and can be potentially transferred across the placenta to the fetus [109, 114]. These studies show that NPs can enter the embryo through the placenta, which is a natural barrier for a large variety of organic substances with diverse molecular structures. The NPs, which appear in the maternal body during pregnancy, can cross the placental barrier and may even cause developmental deform‐ ities. Recently, we demonstrated that CdS-MD nanoparticles were embryotoxics in a chicken embryo model (Figure 5) [42]. The nature of the observed abnormalities suggests that these effects could be directly associated with concentration. Therefore, according to the observed effects, the prolonged accumulation of quantum dots in the maternal organism may increase the risk of adverse effects on embryo development.

The liver is know to have a high capacity to metabolize and degenerate a multitude of xenobiotics. The binding of proteins plays a key role in delivering xenobiotics from plasma into the liver. In addition, the reticuloendothelial system (RES) can phagocytose particles larger than 100 nm and then transport the particles into the liver in vivo. Metabolism of QDs is yet another understudied aspect of cadmium QDs. The QD cores do not appear to be subject to extensive enzymatic metabolism, but shells and coatings are. The extent of metabolism of the shell and coating becomes critical for toxicity, since they shield the more toxic CdSe or CdTe cores from the intracellular environment. Rather than metabolism per se, degradation of the shell and coatings within the biological environment appear to be more important. QD shells and coatings appear to degrade under photolytic and oxidative conditions, yet we know little about the degradation products or their biological effects, which may regulate release of toxic cadmium cores. The behavior of nanoparticles *in vivo* depend on properties such as the

nanosize (surface area and size distribution), chemical composition (purity, crystallinity, electronic property, etc.), surface structure (surface reactivity, surface groups, inorganic or organic coatings, etc.), solubility, shape, and aggregation [115]. It has been suggested that the metabolic paths of QD nanoparticles are closely correlated to their aggregation states, and three metabolic paths were disclosed after intravenous injection: 1) the QDs that maintained their original nanosize without binding *in vivo* were rapidly excreted via the kidney; 2) some QDs binding to proteins were translocated to the liver and were excreted with feces; 3) a small

**Figure 5.** Photographs of 72 h-old chick embryos treated with CdS/Maltodextrin. Observed alterations were dose de‐

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pendent.

also visualize the distribution and accumulation of nanoplexes in brain [107]. Such primary results offer a basis for the development of novel QDs with targeting molecules and drugs, which could enhance drug delivery efficacy across the BBB and facilitate the uptake of the QDdrugs in the brain. Despite the encouraging results on the use of QDs for drug delivery across the BBB, there is still serious concern regarding QDs toxicity *in vivo,* which mainly originates from the intrinsic, potentially toxic nature of the semiconductor materials themselves.

On the other hand, the placental barrier protects the embryo from various chemical agents and other foreign substances in the body. However, the passage of xenobiotic molecules through the placental barrier is not completely prevented, and drugs may affect fetal cell proliferation, embryonic growth, and organ formation [108]. Knowledge regarding embryotoxicity is of great importance because it is a necessary part of the toxicological profile that must be established for any new biologically active substance relevant to human safety. There are many studies in this field [109-111], but the detailed effects of NPs still pose many questions. A decrease in embryonic weight after QDs injection on the sixth day of embryogenesis (CdSe/ZnS QDs, 9.6%; CdT QDs, 6.2%) has been reported, and no embryotoxic or teratogenic effects were observed at any stage of embryogenesis [112]. Others have shown that NPs (nSP and TiO2s 70 nm and 35 nm in diameter, respectively) can cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring [113]. They showed that the NPs were found in the placenta, fetal liver, and fetal brain. Other authors argue that some NPs (CdSe and CdTe/CdS) in different sizes, at different dosages, and with different outer capping materials can increase the rate of early-stage blastocyst death in mice and can be potentially transferred across the placenta to the fetus [109, 114]. These studies show that NPs can enter the embryo through the placenta, which is a natural barrier for a large variety of organic substances with diverse molecular structures. The NPs, which appear in the maternal body during pregnancy, can cross the placental barrier and may even cause developmental deform‐ ities. Recently, we demonstrated that CdS-MD nanoparticles were embryotoxics in a chicken embryo model (Figure 5) [42]. The nature of the observed abnormalities suggests that these effects could be directly associated with concentration. Therefore, according to the observed effects, the prolonged accumulation of quantum dots in the maternal organism may increase

The liver is know to have a high capacity to metabolize and degenerate a multitude of xenobiotics. The binding of proteins plays a key role in delivering xenobiotics from plasma into the liver. In addition, the reticuloendothelial system (RES) can phagocytose particles larger than 100 nm and then transport the particles into the liver in vivo. Metabolism of QDs is yet another understudied aspect of cadmium QDs. The QD cores do not appear to be subject to extensive enzymatic metabolism, but shells and coatings are. The extent of metabolism of the shell and coating becomes critical for toxicity, since they shield the more toxic CdSe or CdTe cores from the intracellular environment. Rather than metabolism per se, degradation of the shell and coatings within the biological environment appear to be more important. QD shells and coatings appear to degrade under photolytic and oxidative conditions, yet we know little about the degradation products or their biological effects, which may regulate release of toxic cadmium cores. The behavior of nanoparticles *in vivo* depend on properties such as the

the risk of adverse effects on embryo development.

484 Application of Nanotechnology in Drug Delivery

**Figure 5.** Photographs of 72 h-old chick embryos treated with CdS/Maltodextrin. Observed alterations were dose de‐ pendent.

nanosize (surface area and size distribution), chemical composition (purity, crystallinity, electronic property, etc.), surface structure (surface reactivity, surface groups, inorganic or organic coatings, etc.), solubility, shape, and aggregation [115]. It has been suggested that the metabolic paths of QD nanoparticles are closely correlated to their aggregation states, and three metabolic paths were disclosed after intravenous injection: 1) the QDs that maintained their original nanosize without binding *in vivo* were rapidly excreted via the kidney; 2) some QDs binding to proteins were translocated to the liver and were excreted with feces; 3) a small fraction of the QDs aggregated to larger particles and were retained in liver tissue for a long time [116].

**7. Strategies for safe drug delivery using cadmium-containing quantum**

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Discussion regarding the toxicity of cadmium-containing QDs can be somewhat confusing because of the diversity QDs being synthesized. Besides and as we have mentioned before, we have to consider that not all QDs are alike. Each individual type of QD possesses its own unique physicochemical properties, which in turn determine its potential toxicity or lack thereof [124]. In general, there are discrepancies in the current literature regarding the toxicity of QDs and these can be attributed to several factors: the lack of toxicology-based studies, the variety of QD dosage/exposure concentrations reported in the literature, and the widely varying

Up to date, toxicity studies have been conducted on a variety of both human and non-human cells and cell lines; research has been focused on *in vitro* assays of cytotoxicity [125-131]. *In vitro* studies are very important and can serve as background data to inform the design of *in vivo* studies but, on their own, they provide an insufficient basis for a complete risk assessment. Administration of QDs in animal models has revealed that QDs induce: (1) accumulation of QDs in specific organs [23, 132, 133], (2) excretion in urine, bilis and feces [134], (3) toxicity in selective organs [135], (4) embryotoxicity [109, 111], and (5) oxidative damage [136-138]. Importantly, and a potential source of confusion in assessing QD toxicity, the latter depends on multiple factors derived from both individual QD physicochemical properties and envi‐ ronmental conditions: QD size, charge, concentration, outer coating bioactivity (capping material, functional groups), and oxidative, photolytic, and mechanical stability have each been shown to be determining toxicity factors. Therefore, all these aspects should be extended to examine alternate QD formulations, compositions, and shapes to help facilitate any future generalizations regarding size thresholds in the regulatory context. There are only a few studies specifically designed for toxicological assessment (e.g., dose, duration, frequency of exposure, mechanisms of action). Many of the studies, from which QD toxicity information is derived, it has been cited in reference to it were performed by nanotechnology researchers rather than toxicologists or health scientists. It is therefore difficult to extrapolate the results of such studies in order to reach any conclusions regarding the health and safety of QDs. Nonetheless, these studies may provide important insights that will be useful in guiding the

The wealth of data accumulated from QD toxicity studies is an invaluable asset that should be exploited to design appropriate methodologies to further assess the toxicity of novel QDs. Researchers often neglect to carry out a comprehensive characterization of QDs prior to using them. In our opinion, this step is absolutely necessary, especially before any toxicity screening is started, precisely because the exact property or properties of QDs responsible for said toxicity are still poorly understood. This omission is one of the reasons behind the current state of confusion surrounding this issue. As epigenetic changes may lead to long-term reprogram‐ ming of gene expression long after the initial insult has been removed, results from "nanoe‐ pigenetic" assessments may have important implications on the future use of new

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physicochemical properties of individual QDs.

eventual design of standardized toxicity tests and protocols.

Several studies suggest that the kidneys can remove QDs that are less than 5 nm. It has been observed that, after i.v. administration of CdSe/ZnS-QDs, only 10% and 40% of the injected dose was found in the kidney and liver respectively, suggesting that only a fraction of the total QDs dose passed through this route [117]. Another study quantitatively detected the excretion of QDs in mice feces and urine after i.v. injection of CDSe QDs. The speed of excretion was quicker via feces, and the peak (0.214 ng of Cd) occurred 6–12 h postexposure. The excretion from urine was relatively delayed, and the peak (0.174 ng of Cd) was 24–36 h post-exposure [118]. The elimination of silica-coated CdSe QDs (~5.5 nm) from the body via feces and urine occurred in its totality after 5 days. Yet many reports propose that a portion of the administered QD dose may not be excreted and remains in the tissues. The extent of excretion and the extent of persistence in tissues take on added importance when one considers the potential delivery of QD as a cancer-targeting drug. More comprehensive studies of potential excretion will therefore be critical to QD development as a nanopharmaceutical.

Numerous studies have addressed the cellular level, but these are often difficult to compare due to varied dosing parameters and lack of physicochemical particle characterization. In general, it appears that most QDs examined found ready uptake at the cellular level, primarily via endocytic mechanisms, which depends upon the surface ligands coated over the QD surface [119, 23]. It has been reported that CdSe QDs and CdTe QDs that enter the cell, are visible at the cell surface and in the cytoplasm after a short time. QDs are likely to bind to the cell surface due to their interaction with cell surface glycoproteins and glycolipids [66]. Employing red-and green-emitting cationic QDs, it was found that the CdTe QD distribution was in part dependent on nanoparticle size. In the murine microglial N9 cell line, red cationic QDs (5 nm) were distributed throughout the cytoplasm. In contrast, green and also positively charged QDs (2 nm) were often found in the nucleus of N9 cells upon 1 h of QD exposure [120]. It has been shown that green QDs coated with tri-metoxysilylprolyl urea and acetate groups bind with high affinity in the cell nucleus of mouse 3T3 fibroblasts [121]. In human mammary epithelial tumor cells (MDA-MB-231) green-emitting CdSe/ZnS/SiO2 QDs were packaged in large vesicles found in the perinuclear region [23]. Confocal images have showed that MPAcoated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPOcoated QDs were not found inside cells. These results indicate that cellular uptake of QDs depends upon the hydrodynamic size of the QDs as well as surface coating material [122]. These results strongly suggest that surface coatings can improve cytocompatibility and, consequently, decrease toxicity. Previous studies have shown that long-term exposure of surface coated QDs to their bioenvironment can destabilize the binding strength of the surface molecules, which in turn can yield unprotected QDs inside or outside the cells [123]. Therefore, the stability and binding strength of surface molecules over the QD surface define the cyto‐ compatibility of the QDs and hence their cytotoxicity. Thus, according to previous studies: (1) the surface coating strategy could improve the cytocompatibility of QDs, (2) surface molecules could determine the intracellular uptake and consequent cytotoxicity of QDs, and (3) intra‐ cellular uptake of QDs could depend on their hydrodynamic size.
