Keywords

Nanoliposomes; nanoparticle; animal model; mouse; rat; rabbit; canine; cancer

Nanoparticles’ Use in Pharmacokinetics

Nanoparticles can be used to track the distribution of the nanoparticles themselves and any agent that they may be carrying after injection in animal models of cancer. This has been done in various ways using ex vivo methods, imaging, and measurement of blood levels. Polyethylene glycol (PEG)-encapsulated (PEGylated) liposomes are nanoparticles that improve drug delivery to tumors but do not damage normal tissue. PEGylated encapsulations can carry drugs such as doxorubicin, liposomal belotecan (an analog of camptothecin), or cisplatin. The amount of drug available is determined by calculating the pharmacokinetics. Pharmacokinetics can vary between species, and there is variability between human patients due to the mononuclear phagocyte system, which releases the drug from the liposome package by digesting it. Non-PEGylated nanoparticles release a drug more rapidly than PEGylated nanoparticles do. The pharmacokinetics was measured in various species including mice, rats, dogs, and humans with refractory solid tumors [2].

Measurement of drugs in the bloodstream when the drug is administered with nanoparticles in an animal model of cancer can help determine the biodistribution of the drug along with changes when delivered with nanoparticles. It has been noted that many multidrug-resistant cancers overexpress epidermal growth factor receptor (EGFR). Fourteen athymic nude mice were injected directly into the mammary fat pad with hypoxic MDA-MB-231 breast cancer tumor cells in Matrigel. These mice were the animal model for a study involving EGFR-targeted and EGFR-nontargeted polymer-blend nanoparticles. These PEGylated nanoparticles were loaded with paclitaxel and lonidamine. Paclitaxel is commonly used to treat cancer but has known toxic effects; lonidamine inhibits aerobic glycolysis and induces apoptosis but it, too, has toxic effects (liver toxicity). The combination of these drugs in clinical trials was shown to improve the efficacy of both drugs in a synergistic manner. The goal of this study was to measure levels of both drugs in the tumor and the bloodstream of tumor-bearing mice. Both the targeted and nontargeted nanoparticles had increased biodistribution when compared with the drug solution which was a combination of the drugs (paclitaxel and lonidamine) without nanoparticles. Targeted nanoparticles did improve the pharmacokinetics of lonidamine [3].

Nanoprobes have been used to determine their targeted distribution in a tumor model. Angiogenesis is a critical event enabling tumor growth. Vascular endothelial growth factor (VEGF) and receptor (VEGFR) signaling pathways play an essential and rate-limiting role in promoting tumor-induced angiogenesis. Not only does angiogenesis correlate with the onset of tumor development but also with growth, metastasis, and invasion of tumors. A 100-nm nanoprobe encapsulated with an iodine contrast agent was used to determine tumor vessel permeability using digital mammography [4].

A female Fischer rat was subcutaneously implanted with cells from the 13762 MAT B III rat mammary adenocarcinoma cell line. On Day 7 after inoculation, intravenous injection of a nanoprobe that encapsulated an iodine contrast agent was imaged with the use of clinical digital mammography. The deposition of the nanoprobes usually coincided with regions of high levels of VEGFR-2, which indicate leakier blood vessels. Thus there is enhanced accumulation of nanoprobes in regions of high angiogenic activity [4].

An ex vivo biodistribution model was utilized to track where nanoparticles accumulated in targeted tissue. Lipid-based oil-filled nanoparticles with chelated nickel targeted to EGFR-overexpressing epidermoid carcinoma cells (A431) were injected into nude mice bearing A431 tumors. EGFR is a transmembrane tyrosine kinase receptor that regulates cell proliferation, apoptosis, differentiation, and migration. EGFR is overexpressed in many cancers including cancers of the breast, prostate, ovary, bladder, pancreas, lung, and kidney, as well as glioma, to name a few. In this model, affinity proteins were used to target the tumor. Affinity proteins, composed of 58 amino acid residues bundled into a three-helix scaffold, are small and easy to produce and can have affinity for specific receptors such as EGFR. After the nanoparticles were injected into the nude mouse xenograft model, the mice were euthanized. Sixteen hours after intravenous injection of the nanoparticles, 19% were detected in tumor tissue, 28% in the liver, and 42% in the kidneys. This finding can be used as a tool for targeting drug delivery to EGFR-positive cancers [5].

Biodistribution of nanoparticles can be tracked using various routes of delivery. YIGSR is a pentapeptide laminin-binding site often found on metastatic cancer cells. In the Sarfati et al. study (2011), YIGSR covalently attached to fluorescent nanoparticles (YIGSR-NPs) was injected subretinally into a CB57B/6 mouse model of melanoma. The mouse melanoma model was created by injecting the mice either subretinally or subcutaneously with cells from the M-cherry-labeled B16F10.9 melanoma cell line CRL-6326. The purpose of the subcutaneous injections of tumor cells was to verify that the YIGSR-NPs honed to the tumor; the purpose of the subretinal injection of tumor cells was to duplicate lung metastasis [6]. Mice that had the solid tumor development after subcutaneous injection of melanoma were given YIGSR-NPs intravenously and the targeted nanoparticles honed to the tumor. The control solid tumor group had a greater disposition of nanoparticles in the liver and spleen. Mice with solid melanoma tumors were injected intratumorally with YIGSR-NPs and they were able to retain the nanoparticles after 48 hours. The control group cleared 75% of the nanoparticles from the tumor in 48 hours that were not covalently attached to YIGSR. Mice injected subretinally with the melanoma cells established the lung metastasis and micro- and macro-metastases within 12 days of injection. Following subretinal injection of the YIGSR-NPs, there was a two- to threefold accumulation of nanoparticles in the lungs at 6 and 24 hours compared to controls where accumulation of the noncovalently bound nanoparticles accumulated in the liver and spleen. In addition, this study also suggested that the nanoparticles were excreted through the urine [6].

Quantum dots (QDs) have been used as a marker to track the movement of a single nanoparticle using a dorsal skinfold mouse model. Female BALB/c athymic nude mice were implanted subcutaneously with cells from the human breast cancer cell line KPL-4, which overexpresses HER2. When the tumor reached a volume of 100–200 mm³, a surgical dorsal skin flap was created in the mice by using two sterilized polyvinyl chloride plates containing a window. The movement of the QDs was observed in the dorsal skinfold chamber using a confocal microscope and camera. This technique allowed the observation of a single nanoparticle in circulation, at extravasation, extracellularly, bound to HER2 on the cell membrane, and moving into the perinuclear region. Ultimately the processes of delivery of QD antibody constructs were identified and analyzed and the rate-limiting parameters could be looked at in vivo [7].

Nanoparticles as Diagnostic Imaging Tools

Accurate imaging tools are important in staging, treatment, and prognosis of cancer. Nanoparticles have value as being useful in diagnostic imaging using radioisotopes, combined with fluorescent dyes or antibodies, or having the ability to be active in a magnetic field for magnetic resonance imaging (MRI). Sentinel lymph nodes are used when staging breast cancer and melanoma. The regional lymph nodes can help with prognosis by helping predict the metastatic status of a solid organ tumor. Current methods of detection use lymphoscintigraphy, which involves injecting radiolabeled nanoparticles throughout the tumor followed by nuclear imaging or detection with a gamma probe to track the lymphatic drainage. The most common gamma-emitting tracer used to label nanoparticles is technetium-99. These labeled nanoparticles are called radiocolloids. Metastases in the sentinel lymph nodes indicate a poor prognosis. Another method for detecting sentinel lymph nodes is to use blue dyes and trace the drainage. The combined use of radiocolloids and blue dye improves the detection rate.

There are other optical lymphotropic imaging agents such as the combination of indocyanine green with nanoparticles that fluoresce and are captured using near-infrared (NIR) cameras. In addition, photoacoustic imaging is another optical modality using optically active contrast agents such as gold nanorods or carbon nanotubes used in combination with a pulsed laser and ultrasound.

Another technique used for imaging in the animal model is computed tomography (CT) by lymphotropic tracers. One of the issues discovered was that the amount of gold nanoparticles that are needed would be large, based on pig and mouse studies [8–12]. Mouse models have been used to trace the path of the lymph system as it relates to the primary tumor. In one mouse model the nanoparticulate lymphotropic contrast agent of gold nanoparticles conjugated to anti-CD45 antibodies was injected into the toe of either the front or back paw of a mouse. In this model the contrast agent had a high affinity for the popliteal or the axillary lymph node [9,13].

Nanoablation is a therapy that intensifies the uptake of superparamagnetic iron oxide (SPIO) nanoparticles in both hepatic and nonhepatic tumors compared with standard intravenous dosing. Intratumoral SPIO nanoparticle uptake after nanoablation can be successfully quantified noninvasively with 7-T MRI. Imaging can be used as a method to estimate localized drug delivery after nanoablation. SPIO nanoparticles act as MRI contrast agents because their superparamagnetic core causes more rapid T1 and T2 relaxation of the immediately surrounding tissues [14].

This technique has been applied to Sprague Dawley rats using the N1-S1 rat hepatoma cell line. The animal model was created via percutaneous ultrasound-guided tumor implantation of the N1-S1 hepatoma into the left lateral lobe of the liver. Nanoablation was performed by exposing the left lateral liver lobe via a laparotomy. In addition the left femoral vein was catheterized for injection of DOX-SPIO nanoparticles. Electroporation was performed for 2 minutes after DOX-SPIO nanoparticle injection via a two-pronged electroporation tool. Upon completion of this process, the liver was returned to the abdominal cavity and the incision closed. The rats were euthanized within 10 minutes after nanoablation and immediately returned to the MRI scanner after treatment. With this technique, MRI can be used to measure the uptake of chemotherapeutic drugs conjugated to nanoparticles [14].

Nanoparticles in conjunction with the iron oxide have been used in another animal model for MRI work. A rat model of metastasis was used to test the use of ultrasmall superparamagnetic iron oxide (USPIO) in combination with MRI to detect sentinel lymph nodes. This model used ACI-AXC 9935/Irish rats injected in the rear paw with cells from the hepatoma H-4-II E tumor cell line, with expected metastasis to the popliteal and paraaortic lymph node within 3 weeks after injection. After metastasis was established, rats were injected with the USPIO preparation intravenously and MRI was performed. In rats that did not have metastasis, signal was dissipated within 24–48 hours after injection, whereas in the metastases model, the signal did not decrease in this same time frame [9,15].

Another model using nanoparticles for imaging involves gold nanoparticles and angiogenesis. Angiogenesis has also been studied in BALB/cBYJNarl mice by using gold nanoparticle colloids. These colloids can be used for other purposes such as cancer targeting, diagnosis, or radiotherapy. To study diffusion of contrast agents through vessel leakage, these mice were injected with the mouse colon carcinoma cell line CT-26 subcutaneously in the thigh. This cell line is known to be highly metastatic. After approximately 7 days, when the tumors reached 100–120 mm³, and again after approximately 26 days, when the tumors reached 1000–1200 mm³, the mice were injected through the tail vein with PEG-Au nanoparticles. These gold nanoparticles behave like hydrophilic contrast agents and localize in the tumor after leaking out of the microvessels using enhanced retention and permeation effect which was visualized using microradiology [8].

Imaging using nanotechnology and angiogenesis has utilized lipid nanobubbles as a contrast agent. When tumors induce angiogenesis, the blood vessels are leaky due to large pore cutoff sizes and large fenestrations. These defects in the blood vessels created through angiogenesis allow for delivery of drugs and gene carriers to the tumor tissue, known as the enhanced permeability and retention (EPR) effect. Lipid nanobubbles that were used as a contrast agent for ultrasound were fabricated and evaluated. The nanobubbles were <450 nm. Mouse prostatic RM-1 cancer cells were subcutaneously injected on the dorsal scapular area in BALB/c athymic nude mice and allowed to grow to a diameter of 1.2 cm. Nanobubbles were injected intravenously via the tail vein. The tumor was then imaged via ultrasound for 1.5 hours on anesthetized mice. The nanobubbles improved contrast enhancement for approximately an hour. The red fluorescently dyed nanobubbles were also viewed ex vivo using a confocal laser scanning microscope. The nanobubbles were injected into the tail vein of tumor-bearing mice and after perfusion of the heart with saline, the tumors and muscle of the thigh were removed and viewed with the use of a confocal microscope. The nanobubbles were present in the intracellular spaces in the tumors but were not readily evident in the muscle sections, indicating passive targeting of the tumor [16].

Combining different imaging modalities can increase the sensitivity of diagnosis and perhaps the treatment of cancer. QDs are small semiconductor nanocrystals with a narrow emission spectrum and high intensity of fluorescence; they are often smaller than 100 nm. QDs are typically composed of cadmium selenide (CdSe) [17]. QDs were used recently for in vivo imaging such as sentinel lymph node mapping and embryo development, tumor angiogenesis, and tracking of metastasis. The NIR region of 700–900 nm allows biomolecules to reach a minimum absorbance for in vivo optical imaging. Arginine-glycine-aspartic acid (RGD) peptide-conjugated NIR QDs can be used for tumor vasculature targeting and imaging in living mice. A combination of near-infrared fluorescent (NIRF) imaging, positron emission tomography (PET), and QDs can provide a sensitive and quantitative assessment of the pharmacokinetics and tumor-targeting efficacy of the NIRF QDs. This may lead to the development of fluorescence-guided surgery and other clinical applications. The nude mouse model was used by injecting the mice subcutaneously with U87MG human glioblastoma cells in the flank. The mice were imaged after ⁶⁴Cu-labeled cell adhesion molecule RGD peptide-conjugated QD was injected intravenously. The mice were then imaged with use of micro-PET in vivo, and harvested tumors were imaged ex vivo. The RGD-QD combination targeted the tumor vasculature with minimal extravasation and this dual-function probe could reduce toxicity and overcome tissue penetration limitations of optical imaging [18]. QDs labeled with RGD peptide sequence have been used to image glioblastoma tumors implanted in mice [19].

Knowing the tumor boundaries can be very useful in cancer treatment. Nanoparticles can assist in better defining the boundary of a solid tumor. Oral squamous cell carcinoma is a common head and neck tumor that is typically treated surgically. The ability to define the boundary of the tumor for surgical removal can improve survival rate and quality of life. Oral squamous carcinoma animal model was developed by injecting female BALB/c nude mice subcutaneously in the cheek with cells from the human buccal squamous cell carcinoma cell line BcaCD885. Oral squamous cell carcinoma tumors highly express EGFR, and this was targeted by EGFR monoclonal antibody–conjugated QDs injected intravenously into the mice. Images were maximal between 15 minutes and 6 hours after injection of the conjugated QDs. Images were captured with use of a confocal microscope [20].

Nanoparticles have been used with confocal microscope technology to enhance the ability to visualize tumor cells. QDs linked to alpha-fetoprotein (AFP) antibody (QDs-Anti-AFP) were used as a marker for hepatocellular carcinoma. QDs may be used as an alternative to organic immunoflourescent probes for cancer detection due to their high sensitivity and specificity for cancer cells. Nude mice were implanted subcutaneously with cells from the HCCLM6 human hepatocellular carcinoma cell line. Tumors were allowed to reach 0.5–1 cm in diameter, and then QDs-Anti-AFP probes were injected intravenously. The mice were euthanized and the tissues were examined with use of a confocal fluorescence microscope. This method allowed active tumor targeting and spectroscopic hepatic carcinoma imaging [21].

Image-guided radiotherapy enhances precise delivery of radiation to tumor tissue. A liquid tissue marker that serves as a fixed area (fiducial) for multiple dosing was used in the following study. This liquid marker was made of nanogel and due to its high radiopacity allows for marker-based image guidance in 2D and 3D X-ray-based imaging during radiotherapy. A canine cancer patient with a spontaneous subcutaneous mast cell tumor was used as a model for evaluating the efficacy of a nanogel marker. The nanogel consisting of an acylated derivative of sucrose, polylactic acid, and ethanol plus acrylamide-coated AuNPs was injected intratumorally while the dog was under general anesthesia. Approximately 24 hours after the nanogel injection, a CT scan of the tumor/nanogel region was performed. This was followed by radiation therapy consisting of 4 fractions of 6 Gy radiation over a 16-day period. Radiation therapy was delivered based on the position of the nanogel. This study concluded the nanogel remained constant in size and volume over the time of the treatment. It continued to retain its 3D shape and remained immobilized throughout the course of treatment despite the canine patient exercising or resting, with no side effects noted. This nanogel represents a liquid fiducial marker with high contrast for 2D X-ray imaging. By injecting the gel into the tumor, the AuNP may enhance the effect of the radiation therapy. Tumor tracking in radiotherapy may improve treatment outcomes through the production of a more precise delivery method [22].

Imaging of the Thomsen–Friedenreich (TF) antigen, which is often overexpressed in colorectal cancer, was performed in the rat model. A nanobeacon was developed that would allow visualization of the TF antigen with use of a FL microendoscope. HCT116 cells were injected into the descending colon of 8- to 10-week-old female athymic nude rats (rnu/rnu, homozygous), after which a white light colonoscopy and then fluorescent endoscopy were performed. This nanobeacon allowed visualization of the tumor and may be used to track tumor regression after treatment through fluorescence colonoscopy [23].

In addition to being a useful therapeutic agent, nanotechnology can be a used as a diagnostic tool. Nanoliposomes can function as carriers of radionuclides for targeting solid tumor. By encapsulating therapeutic radionuclides for internal targeted radiotherapy, nanoliposomes can act as a carrier system. Radiotherapeutics (¹⁸⁸Re-liposome) and radiochemotherapeutics (¹⁸⁸Re-DXR-liposome) administered intravenously to nude mice bearing subcutaneous human HT-29 colorectal adenocarcinoma xenografts provide not only a tool for signal photon-emission computed tomography (SPECT) imaging but also a drug carrier for treating HT-29 solid tumor. Micro-SPECT imaging was performed within 48 hours of injection of ¹⁸⁸Re-(DXR)-liposomes. The ¹⁸⁸Re-DXR-liposome accumulated in the liver, spleen, and tumor. In addition tumor uptake could be clearly seen over time. This formulation provides a useful diagnostic tool and delivery system to the tumor, liver, and spleen [24].

Nanoparticles as a Theranostic Tool

The combination of diagnostic imaging and therapy is called theranostic. Theranostics has an increasingly important role in personalized cancer medicine. One goal is to improve solubility of functional agents, protect them from premature degradation, prolong blood circulation, and enhance tumor accumulation [25].

The uses of nanoparticles for theranostic purposes have been done in animal models of cancer. PEGylated nanoparticles are often used to prolong the vascular circulation of drugs used to combat cancer. There are concerns, however, associated with administration of PEGylated nanoparticles, namely, immunogenicity, anti-PEG immune response, biocompatibility, and toxic effects associated with chronic administration. PEG-free, porphyrin-based ultrasmall nanoparticles that mimic lipoproteins (PLPs) have been used to integrate both imaging and therapeutic functions. These PLPs are stable in blood circulation and rapidly dissociate once they accumulate in a tumor, allowing for fluorescence imaging and tumor-selective photodynamic reactivity and therapy [25].

Another theranostic nanoparticle is porphysomes, organic optically active nanovesicles formed from porphyrin bilayers. Porphysomes are nontoxic in mice and have highly self-quenched energy. Porphysomes allow the visualization of lymphatic systems with use of photoacoustic tomography. Photothermal therapy utilizes contrast agents that convert light to heat in a targeted manner. When exposed to laser irradiation, the porphysomes’ energy is released thermally, similar to what occurs in gold nanorods [26].

Nude mice were implanted subcutaneously with KB tumors that were grown for 2–3 weeks. Then the mice were injected with bacteriochlorophyll porphysomes via the tail vein for fluorescence imaging. Nude mice with KB cell subcutaneous xenografts that were injected intravenously with porphysomes and 24 hours later, they underwent photothermal therapy with use of a laser. The tumor showed a therapeutic response with increased survival time for the mice after the photothermal ablation. Additionally the mice underwent NIR resonance imaging and tumor temperature was measured using an infrared camera. The tumors fluoresced 2 days after injection due to accumulation of the porphysomes in the tumor [26]. Unlike the PLPs, the porphysomes are known to require PEG to increase the stability and avoid rapid clearance by the reticuloendothelial system (RES) [25].

Biocompatible gum Arabic–stabilized gold nanocrystals (GA-AuNPs) were a theranostic agent that was used as an X-ray contrast agent in a tumor-bearing dog. The data suggested that accumulation of GA-AuNPs reaches a threshold limit within a short period (5 hours). The nanocrystals were retained in the tumor tissue for 24 hours [27].

AuNPs have also been used as theranostic tools. A castrated male client-owned dog was presented with a large ventral cervical mass consisting of a mixture of a thyroid carcinoma and osteosarcoma. Metastasis to the lungs was present at the time of diagnosis. With use of a CT scan, intratumoral injections of GA-AuNP were administered under general anesthesia at seven sites of the tumor for a total volume of 2.1 mL. The dog was anesthetized 24 hours after the nanoparticles were injected, and a second CT scan was performed. Images obtained after treatment revealed a slight increase in Hounsefield units (HU) of contrast enhancement of the cervical mass compared with the preinjection CT scan. Increased HU values indicated uptake in the intratumoral mass of the dog. Noted adverse effects were mild edema and mild fever after injection. At 3 weeks after injection, the dog was euthanized due to progressive clinical signs of local disease. This procedure provides a protocol for multiple-injection of intratumoral nanoparticles to ensure effective accumulation of substantial quantities of nanoparticles within a tumor site to produce contrast enhancement [27].

Additionally in a study by Nurunnabi et al. [28], green graphene quantum dots (GQDs) were tested for theranostic abilities in BALB/c athymic nude mice; GQDs were implanted subcutaneously with KB tumor cells (KERATIN-forming tumor cell line HeLa). At 21 days after tumor injection, in vivo imaging was performed. Although photoluminescent GQDs had accumulated in the liver, heart, spleen, lung, kidneys, and tumor sites after intravenous injection of the QDs, the fluorescence signal was observed only in the tumor site with use of a molecular imaging system. The GQDs were approximately 5 nm in diameter [28].

Nanoparticles as a Treatment Tool

One of the major goals of cancer therapeutics is to kill cancer cells while not damaging normal cells. One way to achieve this is the use of molecularly targeted therapy combined with chemotherapy. Tissue and cell distribution of cancer therapeutic drugs can be controlled by entrapment in submicronic (<1 µm) colloidal systems, otherwise known as nanoparticles. Nanoparticles have been shown to potentially reverse multiple-drug resistance. Some of the desirable characteristics that are needed to deliver therapeutic agents to tumor cells include the ability to overcome drug resistance at the tumor and cellular levels and to ensure appropriate distribution, biotransformation, and clearance of the drug [29].

To investigate nanoparticles for treatment purposes, the nanoparticle should be directed to the location of the tumor. First nonmodified nanoparticles were looked at to see where they were deposited after intravenous injection. Nonmodified nanoparticles in the bloodstream are cleared by fixed macrophages typically found in the liver, spleen, lungs, and bone marrow. This was verified by injecting mice in the tail vein with [¹⁴C]-doxorubicin incorporated into polyisohexylcyanoacrylate nanoparticles. This nanoparticle combination had a higher concentration of doxorubicin in the liver, spleen, and lungs compared with concentrations in mice given free doxorubicin, which resulted in higher levels in the heart and kidneys [29,30]. In the metastases model of C57BL/6 mice injected intravenously with reticulosarcoma cell line M 5076, doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles increased the antitumoral cytotoxic activity in the liver [31].

Similar to the use of polyisohexlcyanoacrylate as a carrier for doxorubicin, nanoparticles can provide yet another carrier-mediated drug targeting system for tumors. To counter the fast removal of particles from the circulation by the RES, nanosized particles combined with a hydrophilic surface can delay RES uptake. CD1-Nu mice were implanted subcutaneously with cells from the pancreatic human cell line PANC-1. Once the tumor size reached 75 mm³, nude mice were injected intravenously with doxorubicin (DXR)-loaded targeted hyaluronan liposomes, tHA-LIP-DXR on Days 0, 7, and 14. By Day 32, the response of the animals treated with tHA-LIP-DXR indicated tumor volumes significantly smaller than that at initiation of treatment. Moreover, some mice were tumor-free [32].

Liposomal delivery systems provide modified techniques for improved pharmacokinetic and safety profiles of cytotoxic drugs. The antitumor activity of a nanoliposomal formulation of irinotecan (nal-IRI) can achieve greater intratumoral levels of the prodrug irinotecan and its active metabolite SN-38 compared with free irinotecan. Human colorectal adenocarcinoma HT-29 cells were injected subcutaneously into the right flank of NOD/SCID mice. After the desired tumor volume was achieved, treatments with control, free irinotecan, and nal-IRI were injected intravenously weekly for 4 weeks. Thus the extended exposure of tumor cells to SN-38, which is achieved by nal-IRI, can contribute toward enhanced cytotoxicity compared with free irinotecan. Liposomal encapsulation of irinotecan can safely improve its antitumor activity in preclinical models by enhancing accumulation of its active metabolite within the tumor microenvironment [33].

Nanoliposomal irinotecan has been used as treatment in orthotopic brain tumor models. Chemotherapy has proven to be particularly challenging due to the blood–brain barrier (BBB) and its associated low permeability to a wide variety of drugs. Irinotecan, a widely used cancer chemotherapeutic, displays antitumor activity against various cancer types, including malignant gliomas. Irinotecan has a complex metabolic profile that may limit its ability to provide adequate concentration at the tumor site. Irinotecan is mainly a prodrug, dependent upon conversion to SN-38 by carboxylesterases for optimal anticancer activity [34].

One example of the use of nanoliposomal irinotecan was in athymic nude rats which were injected intracranially with cells from the U87MG glioblastoma multiforme cell line. At 5 days after tumor cell implantation, nanoliposomal irinotecan was administered intravenously. At the end of the study, at Day 100 after tumor implantation, the surviving animals were euthanized and necropsied. The results indicated that nanoliposomal irinotecan was associated with prolonged tumor accumulation/retention and increased survival duration. Furthermore, nanoliposomal delivery of irinotecan produced very high concentrations and prolonged exposure of irinotecan within brain tumors, which was consequently associated with increased intratumoral SN-38 levels. Nanoliposomal irinotecan accumulates progressively to high levels within brain tumors by utilizing the EPR effect of angiogenesis, followed by intratumoral conversion to SN-38. These findings suggest that nanoliposomal irinotecan administered systemically provides significant pharmacologic advantages and may be an efficacious therapy for brain tumors [34].

Various liposomal packaging systems have been developed as primary carriers to avoid phagocytosis and to circulate longer in the blood. An example is folate receptor (FR)-targeted liposomal oridonin, otherwise known as FR-targeted liposomal ORI or F-L-ORI. Tumor-bearing mice were developed by inoculating a suspension of human hepatocellular carcinoma HepG2 cells subcutaneously in athymic nude BALB/c mice. Five days after inoculation, intravenous F-L-ORI was administered. The results indicated that F-L-ORI had produced an antitumor effect in vivo against HepG2 cells [35].

Some tumors are known to have a high number of folic acid receptors. Female BALB/c mice were injected in the footpad with cells from the murine lung carcinoma cell line, M109R-HiFR. This cell line is known to express folic acid at a high level. FR-targeted liposomes that were loaded with doxorubicin were exposed to the M109R-HiFR cells in vitro and then injected into the mice. These cells exhibited less tumor growth than did untreated cells [36,37]. Similarly, FR-targeted lipid nanoparticles loaded with lipophilic paclitaxel was injected into tumor-bearing mice. Paclitaxel is typically formulated in Cremophor EL, which has several adverse effects including nephrotoxicity and hypersensitivity. Female BALB/c mice were injected subcutaneously into the flank with M109 cells and then injected intraperitoneally with lipid FR-targeted nanoparticles with paclitaxel. The tumor growth rate for the mice that received the targeted nanoparticles was less than that for the mice that received paclitaxel in Cremophor EL [38].

There are various nanoliposomal formulations that are effective in treating cancer. Nanoscale ceramide liposomes can be loaded with a variety of anticancer compounds to create a combination therapy against tumors. C6-ceramide is a sphingolipid metabolite that causes cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced. To create the animal model, BALB/c athymic nude mice were irradiated (600 cGy) 1 day before inoculation with cells from the chronic lymphocytic leukemia (CLL) cell line, JVM3. Cells were injected subcutaneously into the right flank of the mice. Treatment began approximately 2 weeks after inoculation. Mice were then treated with C6-ceramide nanoliposomes intravenously, which preferentially inhibit the altered metabolism of glucose in leukemic cells via downregulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), resulting in induction of necrotic cell death [39].

Bilateral human hepatocellular carcinoma tumor xenografts were established in athymic nude mice by subcutaneous injection of SK-HEP-1 cells. One week after tumor cell inoculation, C6-ceramide nanoliposomes were injected intravenously. Administration of nanoliposomal C6-ceramide restricted angiogenesis and induced tumor cell apoptosis, thereby preventing the growth of human hepatocellular SK-HEP-1 tumor xenografts in athymic nude mice [40].

Nanoliposomal C6-ceramide in combination with vinblastine has been used to enhance cell death through apoptosis. Nanoliposomal C6-ceramide is an autophagy inducer, and vinblastine is an autophagy maturation inhibitor. Autophagy is the recycling of cellular proteins and organelles during periods of starvation and is used for survival of the cell. Autophagy is also used to remove damaged organelles and long-lived protein. During solid tumor development, the cancer cells are rapidly multiplying and need nutrients to survive. Autophagy during this phase with poor vascularization allows the solid tumor during the progressive phase to survive. To study this mechanism, athymic nude mice were injected subcutaneously in the flank with cells from the human colon adenocarcinoma cell line LS174T. The nanoliposomal C6-ceramide was injected intravenously via the tail vein followed by vinblastine 15 minutes later. This combination treatment resulted in suppression of tumor growth when compared to nanoliposomal C6-ceramide and vinblastine alone [41].

Another animal model for the use of ceramide for treatment of leukemia using nanoliposomes was the Fischer 344 rat. Ceramide as noted previously has been recognized as an antiproliferative and proapoptotic sphingolipid metabolite. Cells from the RNK-16 in vivo T-cell large granular lymphocyte (LGL) leukemic cell line were intraperitoneally transplanted into Fischer 344 rats. Five weeks after inoculation, leukemic rats were treated with C6-ceramide nanoliposomes via tail vein injections for 6 weeks. The C6-ceramide nanoliposome–treated group exhibited greater survivability than the untreated and ghost nanoliposome (no ceramide) group. More importantly, some of the rats treated with nanoliposomal C6-ceramide had maintained normal blood cell counts without circulating immature blood cells, suggesting achievement of complete clinical remission. The responsive rats were euthanized and necropsied at various time points to further evaluate remission. At necropsy, there was resolution of organomegaly, representing a 3- to 10-fold reduction in weight. In addition, these rats had normal levels of LGL cells in the blood, marrow, lymph nodes, and lung as well as normal splenic histology. This study demonstrated that the nanoliposomal delivery of C6-ceramide to rats with LGL leukemia induces significant apoptotic cell death, which may potentially lead to resolution of leukemic cell infiltration [42].

Nanoliposomal ceramide also has useful properties as an effective antipancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization. The use of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a glucosylceramide synthase inhibitor, and gemcitabine, a nucleoside analog, enhances the antitumor activity of C6-ceramide liposome. Cells from the human pancreatic cell line PANC-1 were injected subcutaneously in athymic nude mice followed by intravenous injection of nanoliposomal ceramide with gemcitabine. This combination therapy inhibited growth of the tumor [43].

Nanoliposomal delivery systems have been created by attaching tissue-specific antibodies. Nanoliposomes conjugated to thyroid-stimulating hormone (TSH) bind to the TSH receptor (TSHr) on the surface of thyrocytes. The tumor model to study the ability of nanoliposomes to target the thyroid was developed by injecting follicular thyroid carcinoma (FTC-133 cells) subcutaneously into NOD/SCID mice. In this way, the in vivo anticancer efficacy of gemcitabine, gemcitabine-loaded nanoliposomes, and gemcitabine-loaded TSH-nanoliposomes could be evaluated. Free gemcitabine and both formulations containing gemcitabine nanoliposomes were administered intravenously after the subcutaneous tumor reached the desired volume. Gemcitabine-loaded TSH-nanoliposomes elicited the greatest reduction in tumor growth [44].

Radioactive nanoliposomes are not only beneficial for imaging but have therapeutic applicability as well. The efficacy of radioactive rhenium (¹⁸⁸Re)-labeled nanoliposomes was also studied in a C26 colonic peritoneal carcinomatosis mouse model. The use of ¹⁸⁸Re-liposomes for passively targeted tumor therapy had greater therapeutic effect than did the chemotherapeutic drug 5-fluorouracil (5-FU) in a colonic peritoneal carcinomatosis mouse model. Nanotargeted ¹⁸⁸Re-liposomes administered intravenously have been used for internal radiotherapy of peritoneal carcinomatosis and ascites. BALB/C mice were inoculated intraperitoneally with the C26 murine colon carcinoma cell line. Seven days after tumor cell inoculation, colon peritoneal metastatic carcinoma-bearing mice received an intravenous injection of ¹⁸⁸Re-liposomes, 5-FU, or normal saline. Results demonstrated better therapeutic efficacy by inhibiting the progression of peritoneal tumor growth for mice that received intravenously administered ¹⁸⁸Re-liposomes. In addition, a therapeutic efficacy study exhibited a greater survival time in mice treated with the ¹⁸⁸Re-liposomes. Furthermore, Micro-SPECT/CT images showed high uptake and targeting of ¹⁸⁸Re-liposomes in ascites and in the tumor, liver, and feces in the colon. This provides a useful tool for in vivo and ascites retention in peritoneal carcinomatosis mice. The information suggests that ¹⁸⁸Re-liposomes have potential benefits for imaging along with the treatment of tumor nodules [45].

Nanoliposomes have a myriad of possible compositions making them a flexible solution to deliver drugs to specific targeted tissues such as tumors. Antitumor efficacy of both nontargeted and HER2-targeted nanoliposome formulations of topotecan was studied in prostate and breast tumor xenografts. Nanoliposomal topotecan (nLs-TPT) that was stabilized with use of sucrose octasulfate was shown to have significant antitumor activity against a DU145 human prostate tumor xenograft model. Nude mice implanted with DU145 prostate cancer cells subcutaneously in the upper back were given six doses of nLs-TPT beginning at 6 days (mean tumor volume of 150 mm³) after tumor implantation; nLs-TPT was administered every 4 days via the tail vein. The results indicated a substantial regression and even complete cures in 5 of 10 mice in the nLs-TPT group. However, weight loss was noted if the number of doses was increased from 4 to 6. Lastly, mice implanted with cells from the human breast cancer cell line BT474 that overexpressed HER2 did not have complete regression of tumor and experienced significant weight loss during drug therapy beyond 5 doses [46].

Docetaxel formulations have been developed for the purpose of improving its solubility. Docetaxel is not readily water-soluble and is often dissolved in polysorbate 80, which is known to cause hypersensitivity, febrile neutropenia, nail changes, and other adverse effects. Docetaxel is a chemotherapeutic drug similar to paclitaxel used to treat various cancers such as cancers of the breast, stomach, head and neck, and prostate, as well as nonsmall cell lung cancer. Docetaxel-loaded PEG copolymer micelles and docetaxel-loaded PEG copolymer thermosensitive hydrogel were used to treat tumors in the mouse model. Female BALB/c mice were injected subcutaneously with 4T1 mouse breast cancer cells, and tumors were allowed to reach 20–100 mm³ before treatment was initiated. The docetaxel micelle was delivered intravenously every third day, and the docetaxel hydrogel was injected directly into the tumor. The docetaxel micelles had a better outcome on survivability than the docetaxel hydrogel group did [47].

Paclitaxel is a known hydrophobic drug, therefore making it insoluble in water. Typically paclitaxel is solubilized in a combination of ethanol and a polyethoxylated castor oil called Cremophor. Cremophor is known to cause allergic reactions and patients are often given antihistamines and corticosteroids to prevent allergic reactions prior to dosing with paclitaxel [48].

Unmodified paclitaxel, noncovalently mixed with PEGylated hydrophilic carbon clusters (PEG-HCCs), was used as a drug delivery system in mice with orthotopic head and neck tumors. Hydrophilic carbon clusters (HCCs), which are nanoparticles, were covalently modified with polyethylene glycol (PEG-HCCs) and shown to be nontoxic and an effective drug delivery vehicle in nude mice with OSC-19 oral squamous cell carcinoma in the tongue. Paclitaxel mixed with PEG-HCCs (PTX/PEG-HCCs) that were <40 nm was administered intravenously for 12 days after injection of the OSC-19 cells. The tumors were measured twice a week. After 50 days, the tumors treated with the PTX/PEG-HCC significantly decreased tumor volume and increased survival compared to negative controls [48].

Nanotechnology may be used for a vast array of experiments including assisting with the transport of drugs across BBB. Rats have contributed to science as models for various tumor types. Combining tumor therapy with nanotechnology has led to many distinct new approaches for cancer therapy. Polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles were shown to enable the transport of a number of drugs including the antitumor antibiotic doxorubicin (DOX) across the BBB to the brain after intravenous administration to considerably reduce the growth of brain tumors in rats [49].

Malignancies of the central nervous system are resistant to systemic chemotherapy, primarily due to the presence of the BBB, which limits penetration of antineoplastic drugs into brain tumors. The BBB acts as an anatomical and physiological barrier formed by a monolayer of endothelial cells that exhibit specific properties such as intercellular tight junctions, which prevent paracellular transport [49].

To create an intracranial glioblastoma model, glioblastoma homogenated brain tissue was stereotaxically injected into the right lateral ventricle of a Wistar rat brain. DOX bound to polysorbate 80-coated (butyl cyanoacrylate) nanoparticles was injected intravenously for the delivery of drugs to solid tumors. Administration of nanoparticulate DOX was associated with a considerably lower mortality and weight loss when compared with use of the free drug. It could therefore be concluded that DOX toxicity could be decreased by using nanoparticles [49].

The use of nanoliposomes extends beyond simple drug delivery. Cationic nanoliposomes offer a useful tool as a carrier because of their ability to form complexes with negatively charged siRNA. siRNA technology has great potential as a therapeutic modality for targeted gene silencing in cancer treatment [50]. In the Matters et al. study (2009), athymic nude mice were injected subcutaneously into the flank with cells from the human pancreatic cell line, AsPC-1. At 14 days after inoculation of tumor cells, cationic nanoliposomes were used as a delivery vehicle to administer gastrin siRNA intravenously. Results indicated a 25% reduction in tumor size. Because the half-life of siRNAs in the peripheral blood is short, cationic nanoliposomes are a promising vehicle of choice [51].

Cationic nanolipoplexes can function as pulmonary cellular delivery system for siRNA. In the mouse model of metastatic lung cancer induced by the B16F10 skin melanoma cell line injected intravenously, intratracheal administration of the cationic nanolipoplex delivering myeloid cell leukemia sequence 1-specific siRNA (siMcl1) reduced expression in B16F10 cell lines. Reduced formation of melanoma tumor nodules was observed in the lung, demonstrating the utility of cationic nanolipoplexes for pulmonary delivery of therapeutic siRNA for the treatment of lung cancers and possibly for other respiratory diseases. Intratracheal administration of siMcl1 in ECL nanolipoplexes inhibited the growth of B16F10 cells in lung tissues. ECL nanolipoplexes provide a means for delivering protein-specific siRNAs to lung cells in an aerosolized manner that target malignant proteins [52].

The use of lipid nanoparticles with either mRNA or siRNA allows the delivery of nucleic acid to tumors in a programmable manner. Overexpression of tumor suppressor microRNAs (mRNAs) or inhibition of oncogenic mRNAs may have a therapeutic effect for various cancers. Viral delivery systems have been used to carry mRNA and siRNA. Viral delivery systems may have adverse effects associated with toxicity, immunity, or insertions into the genome. siRNAs interfere with the expression of specific genes, whereas mRNAs are RNA fragments that prevent the production of a protein by binding and destroying the messenger RNA that would have produced the protein. The KP mouse model of autochthonous lung adenocarcinoma has shown upregulation of the oncogenic KRAS (Kirsten rat sarcoma) allele and inactivation of the tumor suppressor gene p53. In the KP mouse, tumors were initiated by intranasal inhalation of Adeno-Cre to delete p53 and to activate KRAS. Ten weeks after lung tumor initiation, lipid nanoparticles with mRNA or siRNA were injected intravenously into the mice and significantly delayed tumor progression [53].

Point mutations in the KRAS oncogene commonly occur in human patients with pancreatic cancer. Micelles, which are nanoparticles, were used to encapsulate siRNAs that target the KRAS gene. Female BALB/c athymic nude mice were subcutaneously injected in the flank with cells from the PANC-1 human pancreatic cancer cell line. The nanoparticles were injected into mice intravenously via the tail vein after the tumors reached approximately 100 mm³. After 21 days of treatment with nanoparticles, tumors were significantly smaller than were tumors in the controls [54].

Gold nanoparticles have emerged as a form of functional nanomaterials for the treatment of cancer. A liposome-gold nanoparticle (LiposAU NP) was synthesized that could be cleared through the hepatobiliary and renal route. This agent is also used for photothermal therapy, which causes cell death associated with damaged DNA. Pharmacokinetics of the LiposAU NP was measured in Swiss albino mice. BALB/c nude mice were implanted with cells from the human fibrosarcoma cell line HT1080-fluc2-turboFP. Mice given the combination of LiposAU NP and laser therapy had a complete regression of tumor [55].

The premise behind the use of gold nanoparticles in photothermal therapy is the fact that they can absorb light and be used to heat and ablate tumors. To ensure that nontargeted cells are not damaged during NIR irradiation, the AuNPs are infrared-transparent until they accumulate in the tumor cells, thereby reducing background heating in the blood and increasing specificity. Cells from the A431 squamous carcinoma cell line were injected subcutaneously into the thighs of female athymic nude mice; once the tumors reached 100–150 mm³, the mice received an intravenous injection of AuNPs. The treatment, which included NIR irradiation, resulted in complete tumor ablation with no normal tissue damage. In mice that had been given intratumoral AuNP followed by NIR irradiation, there was 89% tumor ablation [56]. Another use of AuNPs in the mouse model was in combination with radiotherapy. Balb/C mice were injected subcutaneously with syngeneic EMT-6 mammary carcinoma. When the tumors reached 50–90 mm³, the mice were injected intravenously with AuNPs combined with X-ray irradiation, which resulted in eradication of most tumors [57].

Another use of gold nanoparticles as thermal agents is the use of laser-activated nano-thermolysis as a cell-elimination technology (LANTCET). LANTCET is a type of nanobased technique to obliterate tumors. LANTCET locates and damages tumor cells by generating intracellular photothermal bubbles around clusters of gold nanoparticles [58].

A demonstration of LANTCET is as described. A rat sarcoma model was created by injecting polymorphic sarcoma 1 (tumor type M1) cells subcutaneously into the inguinal region of the rat. Gold spherical 30-nm particles conjugated to goat antimouse IgG were applied topically for 40 minutes directly to the tumor surface after the skin was incised and reflected away from the tumor site. Next, laser therapy was performed by using laser pulses with the laser beam directed at the center of the tumor. After 24 hours, the rats were euthanized and the tumors excised for evaluation. The results indicated a nonviable (white) area with the diameter close to that of the laser beam. Using clusters of gold nanoparticles clusters, rather than single gold nanoparticles, greatly improved the selectivity of cell killing and enabled the use of lower laser pulse energy, which was less damaging to normal tissues [58].

High-intensity focused ultrasound (HIFU) combined with phase-shift nanoemulsions (PSNEs) is a useful tool for targeting solid tumors. PSNEs are lipid-coated liquid perfluorocarbon droplets that are less than 200 nm. PSNEs can take advantage of the EPR effect of new blood vessels associated with tumor growth. When PSNEs are vaporized into microbubbles in tumors, they enhance HIFU thermal ablation. New Zealand White rabbits were injected with cells of VX2 carcinoma into the thigh muscle. Tumors were grown for 2 weeks. The rabbits were injected with PSNE in the ear vein, and then 2 hours later, HIFU was performed. HIFU heated the PSNEs and ablated the tumor. PSNEs reduced the time and heat intensity needed to cause thermal ablation of solid tumors, thereby improving the feasibility and efficacy of the treatment [59].

Another method for tumor regression is the use of Carboplatin-Fe@C-loaded chitosan nanoparticles, which possess magnetic targeting and heat production properties. They can target liver cancer tissue by using a static magnetic field along with an alternating magnetic field, raise tumor tissue temperature, and facilitate tumor apoptosis [60].

In a study by Li et al. [60], Rat Walker-256 breast carcinoma cells were inoculated intraperitoneally and grown in donor Sprague Dawley infantile rats; these cells were then implanted via laparotomy into the liver of recipient Sprague Dawley rats. If the tumor grew well, the gastroduodenal artery was isolated via a second laparotomy and catheterized, and Carboplatin-Fe@C-loaded chitosan nanoparticles were injected through the gastroduodenal artery into the hepatic artery. Next, the tumor site on the liver was placed in a 5000-Gs static magnetic field for 30 minutes, and then the abdomen was closed. The rat was then placed in the center coils of a high-frequency induction heater. Survival time was the longest for groups treated with Carboplatin-Fe@C-loaded chitosan nanoparticles, magnetic field, and hyperthermia. Carboplatin-Fe@C-loaded chitosan nanoparticles have demonstrated tumor-targeting capability and precise heating of the target site to reach effective therapeutic temperature without injuring the tissue around the target site [60].

Combining magnetization with a functionalized surface, magnetic nanoparticles can selectively attach to the targeted tissue or cell to achieve their therapeutic role. Shakeri-Zadeh et al. [61] injected BALB/c mice subcutaneously into the flank with cells from the mouse colon tumor cell line CT-26, and the tumor was allowed to grow for 14 days. 5-FU is a fluoropyrimidine antimetabolite that is often used to treat colorectal cancer. 5-FU has a short half-life and is known to have adverse effects such as diarrhea, nausea, mouth sores, and low blood counts. Poly lactic-co-glycolic acid (PLGA)-coated magnetic nanospheres were used to encapsulate the 5-FU. Magnetic drug targeting was performed by exposing the flanks of the mice to a magnetic field of 0.18T after intravenous injection of the nanocapsule in the mice. The PLGA-coated magnetic nanocapsules with 5-FU inhibited tumor growth when exposed to a magnetic field in mice [61].

The development of nanoparticle-integrin antagonist (IA) as a method of drug delivery is another use of nanotechnology. Therapeutic efficacy of polymerized nanoparticles IA complex that targeted tumor neovasculature was performed by using mice bearing M21-L murine melanoma tumors. The mice were given a single systemic injection of nanoparticle/plasmid complex after the tumors reached 400 mm³. All of the mice showed >95% tumor reduction, and the majority showed no evidence of the tumor at 6 days after nanoparticle injection [62]. Another model involved murine CT-26 carcinoma cells that were injected intravenously to induce pulmonary metastases or Balb/C mice that were injected intrasplenically to induce hepatic metastases. The nanoparticle gene complexes were given 10 days after injection of the tumor cells. Little to no visible tumor metastases were observed in the liver or lungs of the mice treated with the nanoparticle complex [62].

Treatment efficacy was tested in a study by Li et al. [63] by using subcutaneously injected K1735-M2 (melanoma tumor) in BALB/c mice and CT-26 (colon adenocarcinoma tumor) in C3H mice. The tumors were allowed to reach 150–200 mm³ before intravenous injection of two nanoparticle complexes to target angiogenesis radiolabeled with Yttrium (⁹⁰Y). The two complexes were the radiolabeled IA-nanoparticle (IA-NP-⁹⁰Y) and radiolabeled monoclonal antibody against murine Flk-1 (anti-Flk-1 MAb)-nanoparticle (anti-Flk-1 Mab-NP-⁹⁰Y) which delayed tumor growth. These radiolabeled nanoparticle complexes were used to target integrin and the VEGFR, which are involved in tumor-induced angiogenesis [63].

Large animal species such as rabbits have been used for nanotechnology projects due to the well-established VX2 tumor model. The VX2 carcinoma (BCRC, Bioresource Collection and Research Center, Taipei, Taiwan) is an anaplastic squamous cell carcinoma derived from a virus-induced papilloma in the wild rabbit but appears as a carcinoma in the domestic species [64]. The VX2 cells may be injected into the organ or muscle of choice to create a tumor at the specific location. The VX2-induced tumor in the leg of a New Zealand White rabbit has been used as a model for nanoparticles incorporating fumagillin, an antimicrobial agent derived from Aspergillus fumagatus, to diminish angiogenesis and reduce VX2 tumor growth in rabbits. Fumagillin nanoparticles may be used to suppress the neovasculature and inhibit VX2 adenocarcinoma development using minute drug doses [65].

The science of nanotechnology has been used to create tiny particles or minicells tagged with anticancer antibodies to specifically attach to cancer cells. Minicells are nonliving, anucleate 400-nm particles that are derived from bacteria. Minicell vectors can act as a package to carry chemotherapeutic drugs. Monastrol is a small molecule that arrests mitosis. EGFR was packaged on a minicell with monastral to deliver the drug in a targeted manner. tMDA-MB-468 is a mammary tumor cell line known to overexpress EGFR. Balb/c nu/nu mice were implanted subcutaneously with human breast adenocarcinoma cells between the shoulder blades. After the tumors grew to ~80 mm³, or after 18 days, the mice were treated with ^EGFRminicells_Monastrol, administered intravenously. The minicells, derived from Salmonella typhimurium, reduced the toxicity associated with chemotherapeutic drugs, allowed reduction of the amount of drug given, and allowed targeting of the tumor [66,67].

In the study by Macdiarmid et al. [66,67] tumor cells were targeted where one arm of the antibody attached to the lipopolysaccharide (LPS) on the minicell surface and the other arm was directed to any tumor cell-surface receptor. Doxorubicin was attached to the minicell (minicell_Dox). Two dogs with spontaneously occurring advanced (stage IV) T-cell non-Hodgkin lymphoma were injected intravenously with anti-canine-CD3 targeted minicells_Dox. Rapid tumor regression was noted. The ^CD3minicells_Dox required less doxorubicin per dose than standard multidrug combination chemotherapy without the minicell packaging system. The minicells were well tolerated without adverse effects or deaths. Due to the bacterial origin of the minicells, caution is advised since systemic administration of bacterial products may elicit an inflammatory response. However, no inflammatory processes were noted in this study despite repeated intravenous administration [66].

Specific delivery of chemotherapeutics to tumors reduces the systemic toxicity often attributed to drugs used to treat cancer. Several virus-based nanoparticles can be used to deliver these chemotherapeutics including bacteriophages and plant viruses. One example is the cowpea mosaic virus (CPMV), which is an RNA virus that replicates in back-eyed peas, tobacco, and quinoa. It has been used in nanobiotechnology to deliver targeted drugs to tumors. The structure of the CPMV is similar to that of poliovirus, rhinovirus, and coxsackievirus. To visualize tumor vascularization, human tumor cells embedded in mesh were implanted into chick chorioallantoic membrane and could be tracked over several days, followed by injection of labeled CPMV [68].

To deliver various agents to tumors, the tumor needed to be targeted. M13 bacteriophage has been used to target the vasculature in mice bearing MDA-MB-435–derived (human breast cancer) tumor xenografts using streptavidin-conjugated QDs administered through the tail vein. Other viral particles that have been used in this manner include cowpea chlorotic mottle virus, canine parvovirus, and MS2 bacteriophage [68,69].

Conclusions

To better understand disease prevention, detection, diagnosis and treatment, living organisms are still required. The use of animal models of cancer and nanoparticles is an ever-changing and dynamic field. As this field continues to evolve, we expect that there will be a trend toward personalized therapies for cancer using engineered nanomaterials. The ability to target specific tissues or trigger drug release at a specific location could reduce the adverse effects of potent chemotherapeutic drugs and allow patients to complete chemotherapeutic regimens that they may not have been able to tolerate previously.

The value of nanotechnology goes beyond its use as a tool for drug delivery. Diagnosis and staging of cancer has the potential to be more accurate using nanotechnology. The use of nanoprobes can be a beneficial prognostic tool. Metastases can be targeted and visualized using nanotechnology. Refinement of the use of nanoparticles and their ability to track tumor cells is continually improving through the utilization of animal models of cancer. Determining if tumor cells have dispersed and traveled to lymph nodes can be more easily detected, thereby allowing physicians to be more knowledgeable about the stage of cancer and determine the best method of treatment.

Nanoparticles can be used as a tool for imaging studies. When seeking to image cancer, contrast agents are often used. An ideal contrast agent would be safe, have minimal adverse effects, have specificity and sensitivity, reach the target quickly, provide high resolution, and be economical.

Treatment of cancer can be more precise using nanoparticles. Tumors are known to have high angiogenic activity and are expected to have more permeable blood vessels, resulting in high intratumoral deposition of nanoscale agents. Furthermore, tumors with leakier blood vessels have the fastest growth rates. Vessel leakiness is strongly associated with the tumor’s environment, growth, and rate of angiogenesis. This known trait for these angiogenic blood vessels can be exploited using nanotechnology that has been tested in the animal model.

Nanoparticles have been designed to hone to particular tissues and receptors on tumor cells. This permits targeted therapy that spares healthy tissue and allows lower levels of chemotherapeutic agents to be used. Chemotherapy often has negative side effects that may be irreversible. Animal models of cancer which improve the efficacy of nanoparticle use will provide improved outcomes for cancer patients.

After cancer treatment, nanoprobes can monitor tumor shrinkage. Nanoparticles can also be used to determine if cancer has returned. Detection of cancer may be possible at earlier stages so that better outcomes can be achieved.

Although this chapter was not an exhaustive compilation of all of the possible animal models of cancer, it provides opportunities for the study of cancer, diagnosis, treatment, and post-treatment evaluation using animal models. Animal models run the gamut of mice, rats, rabbits, and dogs and include spontaneous cancer models, genetically engineered cancer models, and xenografts.