Cytokine and immuno-gene therapy for solid tumors

Despite recent progress in our understanding of cancer biology and in many areas of cancer treatment,the successrate for cancer therapy remains dismal.Immunotherapy for cancer has long been an exciting field for many cancerresearchers due to the possibility to mobilize the body's own immune system to eradicate cancer not only locallybut also systemically.Since its initial discovery,cytokine-based immunotherapy has been vigorously andextensively investigated for cancer treatment due to the perception of it as a relatively easily purifiable,injectableform of cancer treatment agent.However,so far most cytokine-based therapy trials have fallen short ofexpectations.One of main obstacles is the difficulty to achieve therapeutically relevant dosage in patients withoutgenerating excessive normal tissue toxicity.The emergence of novel gene therapy approach to deliver therapeuticcytokine to tumors locally generated great excitement since it has the potential of generating sustained high localconcentration of immunostimulatory cytokine without raising the systemic levels of the cytokines,which isresponsible for most of the observed toxicity.In this review,we will attempt to provide an overview of the field anddiscuss some of the problems associated with cytokine-based immuno-gene therapy and potential solutions.Cellular & Molecular Immunology.2005;2(2):81-91.     

Multi-functional magnetic nanoparticles for magnetic resonance imaging and cancer therapy

We have developed a multi-layer approach for the synthesis of water-dispersible superparamagnetic iron oxide nanoparticles for hyperthermia, magnetic resonance imaging (MRI) and drug delivery applications. In this approach, iron oxide core nanoparticles were obtained by precipitation of iron salts in the presence of ammonia and provided β-cyclodextrin and pluronic polymer (F127) coatings. This formulation (F127250) was highly water dispersible which allowed encapsulation of the anti-cancer drug(s) in β-cyclodextrin and pluronic polymer for sustained drug release. The F127250 formulation has exhibited superior hyperthermia effects over time under alternating magnetic field compared to pure magnetic nanoparticles (MNP) and β-cyclodextrin coated nanoparticles (CD200). Additionally, the improved MRI characteristics were also observed for the F127250 formulation in agar gel and in cisplatin resistant ovarian cancer cells (A12780CP) compared to MNP and CD200 formulations. Furthermore, the drug-loaded formulation of F127250 exhibited many folds of imaging contrast properties. Due to the internalization capacity of the F127250 formulation, its curcumin-loaded formulation (F127250-CUR) exhibited almost equivalent inhibition effects on A2780CP (ovarian), MDA-MB-231 (breast), and PC-3 (prostate) cancer cells even though curcumin release was only 40%. The improved therapeutic effects were verified by examining molecular effects using Western blotting and transmission electron microscopic (TEM) studies. F127250-CUR also exhibited haemocompatibility, suggesting a nanochemo-therapeutic agent for cancer therapy.     

Different muscarinc receptors are involved in the proliferation of murine mammary adenocarcinoma cell lines

We described that two different murine mammary adenocarcinoma cell lines, LM3 and LM2 constitutively expressed muscarinic acetylcholine receptors (mAchR). We here demonstrate, by competitive binding experiments with the tritiated muscarinic antagonist quinuclidinyl benzilate that M2 subtype predominates in both tumor cell lines. Concordantly immunoblotting assays indicate that mAchR exhibit the following order of expression: M2 > M4 > M3 > M1 > M5 in both tumor cell lines. Activation of mAchR with carbachol (CARB) increased proliferation in both tumor cell lines in a concentration dependent manner. In LM3 cells CARB promoted proliferation via M3 receptor activation via inositol 1,4,5-triphosphate and nitric oxide production. CARB-induced LM2 cells proliferation needed both M2 and M1 receptor activation, promoting prostaglandin E2 liberation and arginase catabolism respectively, both of them involved in tumor cell growth.     

Protein modified upconversion nanoparticles for imaging-guided combined photothermal and photodynamic therapy

In this work, we develop a multifunctional nano-platform by coating upconversion nanoparticles (UCNPs) with bovine serum albumin (BSA), obtaining UCNP@BSA nanoparticles with great solubility and stability in physiological environments. Two types of dye molecules, including a photosensitizer, Rose Bengal (RB), and an NIR-absorbing dye, IR825, can be simultaneously loaded into the BSA layer of the UCNP@BSA nanoparticles. In this carefully designed UCNP@BSA-RB&; IR825 system, RB absorbs green light emitted from UCNPs under 980-nm excitation to induce photodynamic cancer cell killing, while IR825 whose absorbance shows no overlap with upconversion excitation and emission wavelengths, offers nanoparticles a strong photothermal perform under 808-nm laser irradiation. Without showing noticeable dark toxicity, the obtained dual-dye loaded nanoparticles are able to kill cancer via combined photothermal and photodynamic therapies, both of which are induced by NIR light with high tissue penetration, by a synergetic manner both in vitro and in vivo. In addition, the intrinsic paramagnetic and optical properties of Gd³⁺-doped UCNPs can further be utilized for in vivo dual modal imaging. Our study suggests that UCNPs with well-designed surface engineering could serve as a multifunctional nano-platform promising in cancer theranostics.     

Theranostic porphyrin dyad nanoparticles for magnetic resonance imaging guided photodynamic therapy

Photodynamic therapy (PDT) is a site-specific treatment of cancer involving the administration of a photosensitizer (PS) followed by the local light activation. Besides efficient PSs, image guidance is essential for precise and safe light delivery to the targeting site, thus improving the therapeutic effectiveness. Herein, we report the fabrication of theranostic porphyrin dyad nanoparticles (TPD NPs) for magnetic resonance imaging (MRI)-guided PDT cancer therapy, where the inner metal free porphyrin functions as a photosensitizer for PDT while the outer Mn-porphyrin serve as an MRI contrast agent. Covalent attachment of porphyrins to TPD NPs avoids premature release during systemic circulation. In addition, TPD NPs (～60 nm) could passively accumulate in tumors and be avidly taken up by tumor cells. The PDT and MRI capabilities of TPD NPs can be conveniently modulated by varying the molar ratio of metal free porphyrin/Mn-porphyrin. At the optimal molar ratio of 40.1%, the total drug loading content is up to 49.8%, 31.3% for metal free porphyrin and 18.5% for Mn-porphyrin. The laser light ablated the tumor completely within 7 days in the presence of TPD NPs and the tumor growth inhibition was 100%. The relaxivities were determined to be 20.58 s⁻¹ mm⁻¹ for TPD NPs, about four times as much as that of Mn-porphyrin (5.16 s⁻¹ mm⁻¹). After 24 h intravenous injection of TPD NPs, MRI images showed that the whole tumor area remained much brighter than surrounding healthy tissue, allowing to guide the laser light to the desired tumor site for photodynamic ablation.     

Age related differences in immunocompetence and incidence of mammary adenocarcinoma in murine mammary tumor virus-infected C3H/Bi mice

In breeder C3H/Bi female mice, infected neonatally by murine mammary tumor virus (MTV), the incidence of spontaneous mammary tumors is greater than 95% between 5 and 9 months of age. In young (2–3 months) female the probability for developing a tumor in the next month is negligible, higher than 80% in mice of middle age (5–6 months) but lower than 4% in aged (10–12 months) females. The age-related changes of some immune functions of spleen cells from these tumor free female mice have been evaluated. While the proliferative capacity of cells to Phytohemagglutinin (PHA) increases, the T cell-dependent antibody response against sheep red blood cells (SRBC) and the antibody-dependent cellular cytotoxicity (ADCC) are significantly decreased in 5–6-month-old mice as compared to the young (2–3 months) female mice.The antibody response against SRBC and the mitogenic response to PHA decline markedly in 10–12-month-old mice but the ADCC increases in this group of mice. In addition, assays with monoclonal anti-Lyt-1 and anti-Lyt-2 antibodies indicate that percentage of Lyt 1⁻ 2⁺ cells (suppressor and cytotoxic T cells) is lower in 10–12-month-old female as compared to 5–6-month-old animals. These results show that the immune alterations observed in 10–12-month-old C3H/Bi mice are not closely associated with an increase in incidence of spontaneous tumors and suggest that a high non-T killer cell activity could protect some of these older C3H/Bi female mice against mammary tumor development.     

Cytotoxicity suppression and cellular uptake enhancement of surface modified magnetic nanoparticles

The aim of this study was to modify the surfaces of superparamagnetic iron oxide nanoparticles (SPION) with pullulan in order to reduce the cytotoxicity and enhance the cellular uptake of the nanoparticles. In this study, we have prepared and characterised the pullulan coated superparamagnetic iron oxide nanoparticles (Pn-SPION) of size around 40-45 nm with magnetite inner core and hydrophilic outer shell of pullulan. We have investigated the effect of cellular uptake of uncoated and Pn-SPION on cell adhesion/viability, cytotoxicity, morphology and cytoskeleton organisation of human fibroblasts. Cell cytotoxicity/adhesion studies of SPIONs on human dermal fibroblasts showed that the particles are toxic and their internalisation resulted in disruption of cytoskeleton organisation of cells. On the other hand, Pn-SPIONs were found to be non-toxic and induced changes in cytoskeleton organisation different from that observed with SPION. Transmission electron microscopy results indicated that the SPION and Pn-SPION were internalised into cells via different mechanisms, thereby suggesting that the particle endocytosis behaviour is dependent on the surface characteristics of the nanoparticles.     

Tumor rejection by gene transfer of the MHC class II transactivator in murine mammary adenocarcinoma cells

The murine mammary adenocarcinoma cell line TS/A is a highly malignant MHC class II-negative tumor. We show that transfection of TS/A cells with the MHC class II transactivator CIITA renders them MHC class II-positive and highly immunogenic in vivo. These cells were fully rejected by 51% of syngeneic recipients and had a significantly lower growth rate in the remaining 49% of animals. This directly correlated to the amount of MHC class II molecules expressed in the transfected tumor. Tumor rejecting animals were protected against rechallenge with the parental TS/A tumor. The rejection required CD4(+) and CD8(+) T cells. CD4(+) T cells were fundamental in the priming phase of the antitumor response. CTL-specific for a peptide of the envelope gp70 of an endogenous ecotropic retrovirus were identified and explained the specificity of the effector mechanism of rejection against the TS/A and the antigenically related C26 carcinoma cells but not against the unrelated gp70-negative syngeneic fibrosarcoma F1F cells. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the antitumor immune response.     

Polyethylene glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting

While successful magnetic tumor targeting of iron oxide nanoparticles has been achieved in a number of models, the rapid blood clearance of magnetically suitable particles by the reticuloendothelial system (RES) limits their availability for targeting. This work aimed to develop a long-circulating magnetic iron oxide nanoparticle (MNP) platform capable of sustained tumor exposure via the circulation and, thus, potentially enhanced magnetic tumor targeting. Aminated, cross-linked starch (DN) and aminosilane (A) coated MNPs were successfully modified with 5 kDa (A5, D5) or 20 kDa (A20, D20) polyethylene glycol (PEG) chains using simple N-Hydroxysuccinimide (NHS) chemistry and characterized. Identical PEG-weight analogues between platforms (A5 & D5, A20 & D20) were similar in size (140-190 nm) and relative PEG labeling (1.5% of surface amines - A5/D5, 0.4% - A20/D20), with all PEG-MNPs possessing magnetization properties suitable for magnetic targeting. Candidate PEG-MNPs were studied in RES simulations in vitro to predict long-circulating character. D5 and D20 performed best showing sustained size stability in cell culture medium at 37 °C and 7 (D20) to 10 (D5) fold less uptake in RAW264.7 macrophages when compared to previously targeted, unmodified starch MNPs (D). Observations in vitro were validated in vivo, with D5 (7.29 h) and D20 (11.75 h) showing much longer half-lives than D (0.12 h). Improved plasma stability enhanced tumor MNP exposure 100 (D5) to 150 (D20) fold as measured by plasma AUC(0-∞). Sustained tumor exposure over 24 h was visually confirmed in a 9L-glioma rat model (12 mg Fe/kg) using magnetic resonance imaging (MRI). Findings indicate that a polyethylene glycol modified, cross-linked starch-coated MNP is a promising platform for enhanced magnetic tumor targeting, warranting further study in tumor models.     

Photosensitizer-conjugated magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging and targeting therapy

A major challenge in nanotechnology and nanomedicine is to integrate tumor targeting, imaging, and selective therapy functions into a small single nanoparticle (<50 nm). Herein, photosensitizer-conjugated magnetic nanoparticles with ～20 nm in diameter were strategically designed and prepared for gastric cancer imaging and therapy. The second generation photosensitizer chlorin e6 (Ce6) was covalently anchored on the surface of magnetic nanoparticles with silane coupling agent. We found that the covalently incorporated Ce6 molecules retained their spectroscopic and functional properties for near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT), and the core magnetic nanoparticles offered the functions of magnetically guided drug delivery and magnetic resonance imaging (MRI). The as-prepared single particle platform is suitable for simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer or other tumors.     

磁性纳米颗粒在癌症诊疗一体化中的应用进展

癌症是21世纪威胁人类健康的三大杀手之一,目前比较有前景的研究方向是关于癌症的早期诊断和高效低毒的治疗方案,纳米技术的兴起给此方面研究带来了新的希望.其中,磁性纳米颗粒由于可以在固定磁场中定位,且具有可在交变磁场振动发热的特性,因此在诊疗一体化方面有显著的效果.随着对磁性纳米颗粒的深入研究,磁性纳米颗粒也必然在癌症治疗方面展现出独特能力.综述了单金属、双金属以及合金纳米颗粒在癌症诊疗一体化中的主要应用及进展,并对磁性纳米技术的应用前景进行了展望.磁性纳米颗粒目前已经得到了较为广泛的应用,而且无论是理论研究还是应用研究都发展得很快,但在磁性纳米颗粒被确认为低毒有效、诊疗俱备之前,磁性纳米颗粒对细胞、组织与器官的毒性、商用及医用标准化等问题还有待继续深入研究.     

Tailored magnetic nanoparticles for optimizing magnetic fluid hyperthermia

Magnetic Fluid Hyperthermia (MFH) is a promising approach towards adjuvant cancer therapy that is based on the localized heating of tumors using the relaxation losses of iron oxide magnetic nanoparticles (MNPs) in alternating magnetic fields (AMF). In this study, we demonstrate optimization of MFH by tailoring MNP size to an applied AMF frequency. Unlike conventional aqueous synthesis routes, we use organic synthesis routes that offer precise control over MNP size (diameter ～10 to 25 nm), size distribution, and phase purity. Furthermore, the particles are successfully transferred to the aqueous phase using a biocompatible amphiphilic polymer, and demonstrate long-term shelf life. A rigorous characterization protocol ensures that the water-stable MNPs meet all the critical requirements: (1) uniform shape and monodispersity, (2) phase purity, (3) stable magnetic properties approaching that of the bulk, (4) colloidal stability, (5) substantial shelf life, and (6) pose no significant in vitro toxicity. Using a dedicated hyperthermia system, we then identified that 16 nm monodisperse MNPs (σ-0.175) respond optimally to our chosen AMF conditions (f = 373 kHz, H? = 14 kA/m); however, with a broader size distribution (σ-0.284) the Specific Loss Power (SLP) decreases by 30%. Finally, we show that these tailored MNPs demonstrate maximum hyperthermia efficiency by reducing viability of Jurkat cells in vitro, suggesting our optimization translates truthfully to cell populations. In summary, we present a way to intrinsically optimize MFH by tailoring the MNPs to any applied AMF, a required precursor to optimize dose and time of treatment.     

PEG-PLA nanoparticles modified with APTEDB peptide for enhanced anti-angiogenic and anti-glioma therapy

Tumor neovasculature and tumor cells dual-targeting chemotherapy can not only destroy the tumor neovasculature, cut off the supply of nutrition and starve the tumor cells, but also directly kill tumor cells, holding great potential in overcoming the drawbacks of anti-angiogenic therapy only and improving the anti-glioma efficacy. In the present study, by taking advantage of the specific expression of fibronectin extra domain B (EDB) on both glioma neovasculature endothelial cells and glioma cells, we constructed EDB-targeted peptide APT_EDB-modified PEG-PLA nanoparticles (APT-NP) for paclitaxel (PTX) loading to enable tumor neovasculature and tumor cells dual-targeting chemotherapy. PTX-loaded APT-NP showed satisfactory encapsulated efficiency, loading capacity and size distribution. In human umbilical vein endothelial cells, APT-NP exhibited significantly elevated cellular accumulation via energy-dependent, caveolae and lipid raft-involved endocytosis, and improved PTX-induced apoptosis therein. Both in vitro tube formation assay and in vivo matrigel angiogenesis analysis confirmed that APT-NP significantly improved the antiangiogenic ability of PTX. In U87MG cells, APT-NP showed elevated cellular internalization and also enhanced the cytotoxicity of the loaded PTX. Following intravenous administration, as shown by both in vivo live animal imaging and tissue distribution analysis, APT-NP achieved a much higher and specific accumulation within the glioma. As a result, APT-NP-PTX exhibited improved anti-glioma efficacy over unmodified nanoparticles and Taxol^® in both subcutaneous and intracranial U87MG xenograft models. These findings collectively indicated that APT_EDB-modified nanoparticles might serve as a promising nanocarrier for tumor cells and neovasculature dual-targeting chemotherapy and hold great potential in improving the efficacy anti-glioma therapy.     

Doxorubicin-loaded polysaccharide nanoparticles suppress the growth of murine colorectal carcinoma and inhibit the metastasis of murine mammary carcinoma in rodent models

As a synergistic drug combination, doxorubicin-loaded cisplatin crosslinked polysaccharide-based nanoparticles (Dex-SA-DOX-CDDP) have demonstrated enhanced antitumor efficacy and reduced systemic toxicity via optimized biodistribution, controlled drug release, prolonged blood circulation, and improved tolerability, compared to the non-crosslinked nanoparticles or free doxorubicin. Herein, we apply the Dex-SA-DOX-CDDP nanoparticles as an efficient antitumor agent to treat colorectal and breast tumors in three different in vivo models, i.e. subcutaneously implanted colorectal carcinoma, dimethylhydrazine-induced autochthonous colorectal carcinoma, and metastatic mammary carcinoma, which more closely simulate the natural milieu of the original tumor with intact pathological and immunological responses. Based on the properties of this combination in higher tumor accumulation and penetrating efficiency, the Dex-SA-DOX-CDDP nanoparticles significantly decreased the tumor sizes in CT26 cell line xenograft tumors compared to control. In addition, the affected animals' lifespan was significantly extended after the Dex-SA-DOX-CDDP treatment, in the autochthonous colon cancer model. Moreover, with the aid of iRGD, Dex-SA-DOX-CDDP could effectively block primary tumor growth and prevent the metastasis of 4T1 murine mammary carcinoma. In conclusion, Dex-SA-DOX-CDDP nanoparticles remarkably inhibit growth of colorectal carcinoma and metastasis of mammary carcinoma in vivo, which provides potential application as a safe and efficient antitumor agent in treatment of these cancers.     

Surface functionalized hollow manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging

Multifunctional hollow manganese oxide nanoparticles (HMON) were produced by a bio-inspired surface functionalization approach, using 3,4-dihydroxy-L-phenylalanine (DOPA) as an adhesive moiety, for cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). Cationic polyethylenimine-DOPA conjugates were stably immobilized onto the surface of HMON due to the strong binding affinity of DOPA to metal oxides, as examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. These nanoparticles were subsequently functionalized with a therapeutic monoclonal antibody, Herceptin, to selectively target cancer cells. Confocal microscopy and MR imaging studies revealed that the surface functionalized HMON enabled the targeted detection of cancer cells in T(1)-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing. These nanomaterials are expected to be widely exploited as multifunctional delivery vehicles for cancer therapy and imaging applications.     

MCP-1 expression as a potential contributor to the high malignancy phenotype of murine mammary adenocarcinoma cells.

The search for mechanisms that regulate tumor progression has motivated the authors' laboratory to establish a unique murine model system, consisting of two lines of DA3 mammary adenocarcinoma cells that were derived originally from a common ancestor but differed in their malignant potential. Studies indicated that the highly malignant phenotype manifested by one of the cell lines (termed Ly-6hi DA3 cells) was associated with high expression of the Ly-6E.1 antigen. To characterize the mechanisms controlling the high malignancy phenotype expressed by Ly-6hi DA3 cells, the study was focussed on the potential contribution of tumor-derived factors to the high malignancy phenotype expressed by these cells. To this end, the expression of CC chemokines, major chemoattractants of monocytes and T cells, by the highly malignant Ly-6hi DA3 cells as compared to the low malignancy Ly-6lo DA3 cells was evaluated. The results indicate that the highly malignant cells express higher levels of factors that induce monocyte migration than the low malignancy cells. Two CC chemokines were shown to be highly produced by Ly-6hi DA3 cells, MIP-1alpha and MCP-1, of which only the latter was shown to contribute to the high migratory activity expressed by the high malignancy Ly-6hi DA3 cells. Since MCP-1 may attract monocytes to tumor sites, these findings suggest that monocyte-derived mediators, such as growth factors or angiogenic cytokines, have pro-malignancy effects that contribute to the high malignancy phenotype expressed by Ly-6hi DA3 cells.     

Captopril-polyethyleneimine conjugate modified gold nanoparticles for co-delivery of drug and gene in anti-angiogenesis breast cancer therapy

Captopril-polyethyleneimine (CP) containing low molecular weight polyethyleneimine and anti-angiogenesis drug captopril conjugated via an amide bond was fabricated to modify gold nanoparticles and complex with siRNA to construct siRNA/CP/GNP complexes for the co-delivery of drug and siRNA in anti-angiogenesis breast cancer therapy. The self-assembled siRNA/CP/GNP complexes exhibited desirable and homogenous particle size, reasonable positive charges and condensation ability, and effective gene-silencing property in vitro. In addition, siRNA/CP/GNP complexes co-delivering captopril and siRNA achieved combined angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor mRNA and protein via different pathways in vitro, as compared to mono-delivery systems. In vivo investigation on nude mice bearing MDA-MB435 tumor xenografts revealed that siRNA/CP/GNP complexes possessed satisfying tumor homing ability and strong antitumor activity. These findings suggested that siRNA/CP/GNP complexes could be an ideal system for simultaneous transfer of drug and siRNA, which might be a new promising strategy for effective breast cancer therapy.     

In vitro characterization of genetically modified embryonic stem cells as a therapy for murine mucopolysaccharidosis type IIIA

The mucopolysaccharidoses (MPS) are lysosomal storage disorders resulting from the impaired catabolism of glycosaminoglycans (GAG). MPS type IIIA patients have dysfunctional sulfamidase enzyme leading to lysosomal storage of the GAG heparan sulfate, severe neurological symptoms including regression in learning, behavioural abnormalities, and premature death. We have engineered mouse D3 embryonic stem (ES) cells to over-express recombinant human sulfamidase. Human sulfamidase was correctly folded and secreted 2h post-labelling as determined by immunoprecipitation and SDS-PAGE analysis of transfected ES cells. Secreted human sulfamidase present in conditioned ES cell media was able to be taken up via mannose-6-phosphate-mediated endocytosis and restored sulfamidase enzyme activity in human MPS IIIA fibroblast cell lines. ES cells underwent directed differentiation to neural precursor populations and were capable of sustained human sulfamidase over-expression at all stages. Additionally, transfected and control cells were proliferative (Ki67+) and expressed several neural markers (nestin, MAP-2, and NF160) as determined by immunofluorescence. These findings suggest the possibility of ES cell-based therapy for the treatment of neurological pathology of MPS IIIA.     

Photosensitizer encapsulated organically modified silica nanoparticles for direct two-photon photodynamic therapy and in vivo functional imaging

Nanoparticle-assisted two-photon imaging and near infrared (NIR) imaging are two important technologies in biophotonics research. In the present paper, organically modified silica (ORMOSIL) nanoparticles encapsulated with either PpIX (protoporphyrin IX) photosensitizers or IR-820 NIR fluorophores were synthesized and optically characterized. Using the former ORMOSIL nanoparticles, we showed: (i) direct excitation of the fluorescence of PpIX through its efficient two-photon absorption in the intracellular environment of tumor cells, and (ii) cytotoxicity towards tumor cells by PpIX under two-photon irradiation. The latter ORMOSIL nanoparticles can be used as efficient NIR fluorescent contrast agents for various types in vivo animal imaging. We applied IR-820 doped ORMOSIL nanoparticles in in vivo brain imaging of mice. We also demonstrated the applications of them to sentinel lymph node (SLN) mapping of mice. Finally, we showed that the nanoprobes could target the subcutaneously xenografted tumor of a mouse for long time observations. ORMOSIL nanoparticles have great potentials for disease diagnosis and clinical therapies.