PET Imaging of TIGIT Expression on Tumor-Infiltrating Lymphocytes.

PURPOSE: Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses. EXPERIMENTAL DESIGN: Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse. RESULTS: Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo-activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry. CONCLUSIONS: This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Enhanced nanoparticle accumulation by tumor-acidity-activatable release of sildenafil to induce vasodilation.

Inefficient nanoparticle accumulation in solid tumors hinders the clinical translation of cancer nanomedicines. Herein, we proposed that sildenafil, a vasodilator ampholyte, could be used to promote nanoparticle accumulation by inducing vasodilation after its tumor acidity-triggered release from the nanocarriers. To confirm this, sildenafil was first encapsulated in a cisplatin-incorporated polymeric micelle. The dense PEG shell of the micelle reduced its endocytosis by cancer cells, which in return resulted in accumulative extracellular release of protonated sildenafil in the acidic tumor microenvironment. The released sildenafil was found to be more effective in enlarging the tumor blood vessels than could be achieved without sildenafil. As a result, we demonstrated considerable improvement in the intratumoral accumulation of the sildenafil-cisplatin co-loaded nanoparticle and its enhanced cancer therapeutic efficacy over the control group. Given the generality of a dense PEG shell and a hydrophobic part in most clinically developed nanomedicines, this work implies the great potential of sildenafil as a simple and universal adjuvant to selectively promote the intratumoral accumulation of nanomedicines, thus improving their clinical translation.

Synchrotron Microbeam Radiation Therapy as a New Approach for the Treatment of Radioresistant Melanoma: Potential Underlying Mechanisms.

PURPOSE: Synchrotron microbeam radiation therapy (MRT) is a method that spatially distributes the x-ray beam into several microbeams of very high dose (peak dose), regularly separated by low-dose intervals (valley dose). MRT selectively spares normal tissues, relative to conventional (uniform broad beam [BB]) radiation therapy. METHODS AND MATERIALS: To evaluate the effect of MRT on radioresistant melanoma, B16-F10 murine melanomas were implanted into mice ears. Tumors were either treated with MRT (407.6 Gy peak; 6.2 Gy valley dose) or uniform BB irradiation (6.2 Gy). RESULTS: MRT induced significantly longer tumor regrowth delay than did BB irradiation. A significant 24% reduction in blood vessel perfusion was observed 5 days after MRT, and the cell proliferation index was significantly lower in melanomas treated by MRT compared with BB. MRT provoked a greater induction of senescence in melanoma cells. Bio-Plex analyses revealed enhanced concentration of monocyte-attracting chemokines in the MRT group: MCP-1 at D5, MIP-1α, MIP-1ß, IL12p40, and RANTES at D9. This was associated with leukocytic infiltration at D9 after MRT, attributed mainly to CD8 T cells, natural killer cells, and macrophages. CONCLUSIONS: In light of its potential to disrupt blood vessels that promote infiltration of the tumor by immune cells and its induction of senescence, MRT could be a new therapeutic approach for radioresistant melanoma.

Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization.

We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows-channel flow and cross-flow-in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.

Melanoma-released exosomes directly activate the mitochondrial apoptotic pathway of CD4+ T cells through their microRNA cargo.

Tumor-derived exosomes (TEX) play an important role in the escape of tumor cells from immune surveillance. However, the details of the mechanism are not fully understood. In this study, the apoptosis of CD4+ T cells increased during treatment with B16-derived exosomes in vitro and in vivo, resulting in accelerated growth of melanoma cells in mice. While the release of exosomes was blocked by disrupting the expression of Rab27a, tumor growth was clearly inhibited, and the percentage of T cells in the tumor environment increased. At the same time, Western blot showed that TEX could increase the activation of caspase-3, caspase-7 and caspase-9 but not caspase-8, down-regulating the anti-apoptotic proteins, including BCL-2, MCL-1 and BCL-xL in CD4+ T cells, and indicating that the TEX activates the mitochondrial apoptotic pathway of CD4+ T cells. These reductions were probably associated with the release of microRNAs, such as miR-690, from TEX to T cells. Our present study reveals for the first time that melanoma-released exosomes may directly activate the mitochondrial apoptotic pathway of CD4+ T cells through their microRNA cargo.

Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells.

In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Antimetastatic pectic polysaccharide from Decalepis hamiltonii; galectin-3 inhibition and immune-modulation.

Melanoma is a malignant neoplasm of major concern because of its high mortality rate and failure of chemotherapy. Previously we have shown that galectin-3, a galactose specific lectin, plays a pivotal role in the initiation of metastasis. It was hypothesized that blocking galectin-3 with galactose rich dietary pectic polymer would inhibit metastasis. The current study analyzes the preventive effect and mode of action of a pectic polymer from Swallow Root (Decalepis hamiltonii) in a preventative study of B16F10 cells lung colonization. Matrix metalloproteinase (MMPs) activity was assayed by zymography. Apoptotic/proliferative markers and cytokines were analyzed by immunoassay. Results indicated ~88% inhibition of lung colonization by SRPP as compared to 60% by CPP and only 7% by GRPP. Further molecular analysis revealed that galectin-3 blockade was associated with down regulation of MMPs and NFκB. Activation of caspases supported the apoptotic effect of SRPP. Infiltration of inflammatory cells into the lung was evidenced by presence of CD11b+ cells and release of the pro-inflammatory cytokine-IL-17, indicating inflammation during the cancer cell colonization process. SRPP enhanced the release of IL-12 that enables the reduction of inflammation. Our data for the first time indicate the effective anti-metastatic effect of SRPP due to both galectin-3 blockade and immunomodulation.

Antitumor and antimetastatic effects of pemetrexed-loaded targeted nanoparticles in B16 bearing mice.

Using nanoparticle delivery for anticancer therapy is a potential new drug modality. We developed a novel gelatinase-stimuli nanoparticle. In this study, we studied the antitumor and antimetastasis effect of pemetrexed-loaded targeted nanoparticles and evaluated the correlation between E-cadherin expression and lung metastasis in subcutaneous xenograft model. Compared with free pemetrexed, pemetrexed-loaded targeted nanoparticles exhibited the best antitumor and antimetastasis efficacy among the four therapeutic groups. The study also indicated that there was an inverse correlation between lung metastasis and E-cadherin expression. These results showed pemetrexed-loaded targeted nanoparticles may be a potent drug for tumor therapy and our preclinical data could provide new direction for clinical therapy of malignant melanoma.

Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells.

Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC.

Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles.

This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

Harnessing medically relevant metals onto water-soluble subphthalocyanines: towards bimodal imaging and theranostics.

Subphthalocyanine (SubPc), a putative fluorophore for optical imaging (OI), was conjugated to chelating ligands (DOTA, DTPA) affording water-soluble conjugates complexed with (non-radioactive) metals relevant to the following medical imaging techniques/therapies: MRI (Gd), PET (Cu, Ga), SPECT (In, Ga, Lu), RIT (Cu, Lu, Y), and NCT (Gd). Magneto-optical properties of ditopic gadolinium species (and optical properties of other metal containing species) were examined (brightness (ε × ΦF) and relaxivity R1) and fluorescence confocal/biphoton microscopy studies were conducted.

Antifibrogenic effects of B16 melanoma-conditioned medium.

BACKGROUND: Some malignant cancers show high levels of local invasiveness by the secretion of soluble factors that can degrade adjacent tissues and suppress surrounding cell growth. We investigated the possibility of treating fibroproliferative scars based on these properties of malignant melanoma. MATERIAL AND METHODS: B16 melanoma-conditioned medium (B16 M-CM) was added to keloid fibroblasts (KFs), and proliferation, migration, and type I collagen production were measured. The cell cycle and signaling pathways were also analyzed. Proteins associated with cell proliferation were measured with Western blot analysis. Animal experiments using a rabbit ear model was performed to confirm the effect of B16 M-CM in vivo. RESULTS: B16 M-CM reduced proliferation, migration, and type I collagen production of KFs. This treatment also increased the number of cells in the subG1 phase and decreased phosphorylation levels of AKT, extracellular signal-regulated kinase1/2, cyclin D1, and c-Myc of KFs. Additionally, B16 M-CM reduced the thickness of rabbit ear scars in the rabbit ear model in vivo. CONCLUSIONS: B16 M-CM can suppress proliferation, migration, and type I collagen production of KFs. In addition, concentrated B16 M-CM reduced scar thickness in the rabbit ear model. The specific proteins involved should be identified in a future study.

Alteronol induces differentiation of melanoma b16-f0 cells.

Alteronol, isolated from microbial mutation strains, has been applied for Chinese and International patents for tumor treatment. The aim of this project study is to investigate characteristics of proliferation and redifferentiation induced by alteronol in B16-F0 mouse melanoma cells. Cell proliferation is determined by tetrazolium salt colorimetric method (MTT assay). Morphological changes were analyzed by using Giemsa staining. The levels of melanin and tyrosinase were measured by spectrophotometry. The mRNA expressions of tyrosinase-related protein Trp1 and Trp2 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The anchorage-independent proliferation of B16-F0 was monitored by the colony formation assay. Tumorigenicity was characterized by an animal model in vivo. The results showed that the proliferation of B16-F0 cells was inhibited by alteronol in a concentration and time dependent manner. All well-known evaluation indexes of melanoma cell differentiation, including morphological changes and tyrosinase activity alteration, were greatly enhanced with the increase of alteronol concentrations. Taken together, the expression of tyrosinase related gene, decreased cell colony formation rate and the tumorigenicity in vivo; all of these revealed that alteronol plays a key role in inducing differentiation and suppressing the proliferation of B16-F0 tumor cells in vitro and in vivo.

Fluorescent-tilmanocept for tumor margin analysis in the mouse model.

BACKGROUND: Dendritic cells (DC) are localized in close proximity to cancer cells in many well-known tumors, and thus maybe a useful target for tumor margin assessment. MATERIALS AND METHODS: [(99m)Tc]- cyanine 7 (Cy7)-tilmanocept was synthesized and in vitro binding assays to bone marrow-derived DC were performed. Fifteen mice, implanted with either 4T1 mouse mammary or K1735 mouse melanoma tumors, were administered 1.0 nmol of [(99m)Tc]-Cy7-tilmanocept via tail vein injection. After fluorescence imaging 1 or 2 h after injection, the tumor, muscle, and blood were assayed for radioactivity to calculate percent-injected dose. Digital images of the tumors after immunohistochemical staining for DC were analyzed to determine DC density. RESULTS: In vitro binding demonstrated subnanomolar affinity of [(99m)Tc]-Cy7-tilmanocept to DC (KA = 0.31 ± 0.11 nM). After administration of [(99m)Tc]-Cy7-tilmanocept, fluorescence imaging showed a 5.5-fold increase in tumor signal as compared with preinjection images and a 3.3-fold difference in fluorescence activity when comparing the tumor with the surgical bed after tumor excision. Immunohistochemical staining analysis demonstrated that DC density positively correlated with tumor percent of injected dose per gram (r = 0.672, P = 0.03), and higher DC density was observed at the periphery versus center of the tumor (186 ± 54 K versus 64 ± 16 K arbitrary units, P = 0.001). CONCLUSIONS: [(99m)Tc]-Cy7-tilmanocept exhibits in vitro and in vivo tumor-specific binding to DC and maybe useful as a tumor margin targeting agent.

Shifting wavelengths of ultraweak photon emissions from dying melanoma cells: their chemical enhancement and blocking are predicted by Cosic's theory of resonant recognition model for macromolecules.

During the first 24 h after removal from incubation, melanoma cells in culture displayed reliable increases in emissions of photons of specific wavelengths during discrete portions of this interval. Applications of specific filters revealed marked and protracted increases in infrared (950 nm) photons about 7 h after removal followed 3 h later by marked and protracted increases in near ultraviolet (370 nm) photon emissions. Specific wavelengths within the visible (400 to 800 nm) peaked 12 to 24 h later. Specific activators or inhibitors for specific wavelengths based upon Cosic's resonant recognition model elicited either enhancement or diminishment of photons at the specific wavelength as predicted. Inhibitors or activators predicted for other wavelengths, even within 10 nm, were less or not effective. There is now evidence for quantitative coupling between the wavelength of photon emissions and intrinsic cellular chemistry. The results are consistent with initial activation of signaling molecules associated with infrared followed about 3 h later by growth and protein-structural factors associated with ultraviolet. The greater-than-expected photon counts compared with raw measures through the various filters, which also function as reflective material to other photons, suggest that photons of different wavelengths might be self-stimulatory and could play a significant role in cell-to-cell communication.

Antagonism by Ganoderma lucidum polysaccharides against the suppression by culture supernatants of B16F10 melanoma cells on macrophage.

It is well-documented that macrophages have the functions to regulate antitumor immune response. Antitumor response can be launched by a series of events, starting with inflammation mediated by monocyte/macrophages, which stimulates natural killer and dendritic cells and finally activates the cytotoxic lymphoid system. Monocytes/macrophages may be the first line of defense in tumors. However, specific and nonspecific immunotherapy for human cancer has shown no success or limited success in clinical trials. Part of the reasons attribute to tumor-derived soluble factors that suppress functions of immune cells or induce apoptosis of these cells, including macrophages. Therefore, antagonism of the suppression on the macrophages is an important goal for tumor immunotherapy. To achieve this purpose, Ganoderma lucidum polysaccharides (Gl-PS) with multiple bioactivities were used on mouse peritoneal macrophages incubating with culture supernatants of B16F10 melanoma cells (B16F10-CS). It was shown that the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages after activation by lipopolysaccharide were suppressed by B16F10-CS, while the suppressions were fully or partially antagonized by Gl-PS. In conclusion, B16F10-CS is suppressive to the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages while Gl-PS had the antagonistic effects against this suppression, suggesting this potential of Gl-PS to facilitate cancer immunotherapy.

Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.

Induced clustered nanoconfinement of superparamagnetic iron oxide in biodegradable nanoparticles enhances transverse relaxivity for targeted theranostics.

PURPOSE: Combined therapeutic and diagnostic agents, "theranostics" are emerging valuable tools for noninvasive imaging and drug delivery. Here, we report on a solid biodegradable multifunctional nanoparticle that combines both features. METHODS: Poly(lactide-co-glycolide) nanoparticles were engineered to confine superparamagnetic iron oxide contrast for magnetic resonance imaging while enabling controlled drug delivery and targeting to specific cells. To achieve this dual modality, fatty acids were used as anchors for surface ligands and for encapsulated iron oxide in the polymer matrix. RESULTS: We demonstrate that fatty acid modified iron oxide prolonged retention of the contrast agent in the polymer matrix during degradative release of drug. Antibody-fatty acid surface modification facilitated cellular targeting and subsequent internalization in cells while inducing clustering of encapsulated fatty-acid modified superparamagnetic iron oxide during particle formulation. This induced clustered confinement led to an aggregation within the nanoparticle and, hence, higher transverse relaxivity, r2 , (294 mM(-1) s(-1) ) compared with nanoparticles without fatty-acid ligands (160 mM(-1) s(-1) ) and higher than commercially available superparamagnetic iron oxide nanoparticles (89 mM(-1) s(-1) ). CONCLUSION: Clustering of superparamagnetic iron oxide in poly(lactide-co-glycolide) did not affect the controlled release of encapsulated drugs such as methotrexate or clodronate and their subsequent pharmacological activity, thus highlighting the full theranostic capability of our system.