Engineering biomolecular systems: Controlling the self-assembly of gelatin to form ultra-small bioactive nanomaterials.

The size of nanocarriers determines the biological property of the materials, especially as it relates to intratumoral distribution. Previous research has shown that sizes of 10-50 nm penetrate deep inside the tumor, resulting in better efficacy. On the other hand, studies have shown that gelatin exhibits excellent biological properties, including compatibility, degradability, and toxicity. Therefore, FDA approved gelatin as a safe material to use as an excipient in injectables. The bottleneck is the nonexistence of smaller-sized gelatin nanoparticles (GNPs) to realize the full potential of these biomaterials. Yet, GNPs with sizes of less than 50 nm have not been reported; the synthetic strategy reported in the literature uses "uncontrolled crosslinking coupled with nanoprecipitation", resulting in larger particle size. We have developed a new method to self-assemble gelatin strands by using an anionic, phosphate-based crosslinker and controlled precipitation. The method we developed produced ultra-small gelatin nanoparticles (GX) of size 10 nm with a high degree of reproducibility, and it was characterized using dynamic light scattering (DLS), Energy-dispersive X-ray spectroscopy (EDS), High-resolution transmission, and scanning electron microscopy (HR-TEM/STEM). We also explored GX as a bioactive platform to encapsulate imaging and therapy agents within the cavity. Interestingly, we were able to encapsulate 2 nm size gold nanoparticles within the void of GX. The versatile nature of the GX particles was further demonstrated by surface functionalizing with larger size gelatin nanoparticles to form core-satellite nanocomposites. Additionally, we studied the tumor penetrability of dye-tagged 10, 50, and 200 nm gelatin nanoparticles. The study showed that smaller size gelatin nanoparticles penetrate deeper tumor regions than larger particles. In general, GX was efficient in penetrating the inner region of the spheroids. The results demonstrate the potential capabilities of ultra-small GX nanoparticles for multi-staged payload delivery, diagnostics, and cancer therapy.

Developmental exposure to silver nanoparticles leads to long term gut dysbiosis and neurobehavioral alterations.

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of consumer products that includes topical wound dressings, coatings for biomedical devices, and food-packaging to extend the shelf-life. Despite their beneficial antimicrobial effects, developmental exposure to such AgNPs may lead to gut dysbiosis and long-term health consequences in exposed offspring. AgNPs can cross the placenta and blood-brain-barrier to translocate in the brain of offspring. The underlying hypothesis tested in the current study was that developmental exposure of male and female mice to AgNPs disrupts the microbiome-gut-brain axis. To examine for such effects, C57BL6 female mice were exposed orally to AgNPs at a dose of 3 mg/kg BW or vehicle control 2 weeks prior to breeding and throughout gestation. Male and female offspring were tested in various mazes that measure different behavioral domains, and the gut microbial profiles were surveyed from 30 through 120 days of age. Our study results suggest that developmental exposure results in increased likelihood of engaging in repetitive behaviors and reductions in resident microglial cells. Echo-MRI results indicate increased body fat in offspring exposed to AgNPs exhibit. Coprobacillus spp., Mucispirillum spp., and Bifidobacterium spp. were reduced, while Prevotella spp., Bacillus spp., Planococcaceae, Staphylococcus spp., Enterococcus spp., and Ruminococcus spp. were increased in those developmentally exposed to NPs. These bacterial changes were linked to behavioral and metabolic alterations. In conclusion, developmental exposure of AgNPs results in long term gut dysbiosis, body fat increase and neurobehavioral alterations in offspring.

A review on RNAi therapy for NSCLC: Opportunities and challenges.

Non-small cell lung cancer (NSCLC) is the primary cause of cancer death worldwide. Despite developments in chemotherapy and targeted therapies, the 5-year survival rate has remained at approximately 16% for the last four decades. NSCLC is a heterogeneous group of tumors that, through mutations and drivers, also demonstrate intra-tumor heterogeneity. Thus, current treatment approaches revolve around targeting these oncogenes, often using small molecule inhibitors and chemotherapeutics. However, the efficacy of these therapies has been crippled by acquired and inherent drug-resistance in the tumor, accompanied by increased therapeutic dosages and subsequent devastating off-target effects for patients. Evidently, there is a critical need for developing treatment methodologies more effective than the current standard of care. Fortunately, RNA interference, particularly small interfering RNA (siRNA), presents an alternative of silencing specific oncogenes to control tumor growth. Although siRNA therapy is subject to rapid degradation and poor internalization in vivo, nanoparticles can serve as nontoxic and efficient delivery vehicles, even introducing combinational delivery of multiple therapeutic agents. Indeed, siRNA-nanoconstructs possess extraordinary potential as an innovative modality to address clinical needs. This state-of-the-art review summarizes the recent advancements in the development of novel nanosystems for delivering siRNA to NSCLC tumors and analyzes the efficacy of representative examples. By illuminating the most promising biomarkers for silencing, we hope to streamline current therapeutic efforts and highlight powerful translational opportunities to combat NSCLC. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Systematic Evaluation of Protein-Based Nanoparticles for Stable Delivery of Small Interfering RNA.

Developing a delivery vehicle to protect siRNA from degradation is a significant challenge. To solve this challenge, researchers attempted to use protein-based nanoparticles to deliver siRNA with limited to moderate success. However, a systematic investigation of comparing the ability of different protein-based nanoparticles as vehicles to deliver siRNA stably within cells is still unavailable. Therefore, in this study we synthesized a library of both non-targeted (proteinsiRNA) nanoparticles (NPs) and targeted (antibody conjugated protein-siRNA) NPs and evaluated ability to stably deliver siRNA in to cells to silence the gene of interest. We investigated nanoparticles of casein, bovine serum albumin, and gelatin for the delivery of siRNA. We synthesized and characterized a total of 12 nanoconjugates; in these conjugates, we either encapsulated, electrostatically attached, or covalently conjugated siRNA. We evaluated the efficiency of attaching siRNA to nanoconjugates, stability, and cellular delivery. The ability of siRNA to silence the protein of interest in cancer cells was also investigated. Among non-targeted conjugates, BSA matrix imparted relatively high stability to siRNA when encapsulated. Among targeted nanoconjugates, gelatin nanoparticles rendered high stability to siRNA upon covalent conjugation to the surface. On comparing with both targeted and non-targeted NPs for release of siRNA within cells, antibody-gelatin-siRNA conjugate exhibited high release and functional activity (down-regulation of target protein levels) within the cells as confirmed by both fluorescence imaging and Western blotting. In summary, our investigations show that targeted gelatin nanoparticles and non-targeted BSA nanoparticles possess high stability and excellent gene suppression capabilities and warrants further studies. We can extend the results from this study to develop stable siRNA delivery vehicles to specifically silence the protein of interest.

Magnetic Iron Nanocubes Effectively Capture Epithelial and Mesenchymal Cancer Cells.

Current methods for capturing circulating tumor cells (CTCs) are based on the overexpression of cytokeratin (CK) or epithelial cell-adhesion molecule (EpCAM) on cancer cells. However, during the process of metastasis, tumor cells undergo epithelial-to-mesenchymal transition (EMT) that can lead to the loss of CK/EpCAM expression. Therefore, it is vital to develop a capturing technique independent of CK/EpCAM expression on the cancer cell. To develop this technique, it is important to identify common secondary oncogenic markers overexpressed on tumor cells before and after EMT. We analyzed the biomarker expression levels in tumor cells, before and after EMT, and found two common proteins-human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) whose levels remained unaffected. So, we synthesized immunomagnetic iron nanocubes covalently conjugated with antibodies of Her2 or EGFR to capture cancer cells irrespective of the EMT status. The nanocubes showed high specificity (6-9-fold) in isolating the cancer cells of interest from a mixture of cells spiked in serum. We characterized the captured cells for identifying their EMT status. Thus, we believe the results presented here would help in the development of novel strategies for capturing both primary and metastatic cancer cells from patients' blood to develop an effective treatment plan.

Plate-Adherent Nanosubstrate for Improved ELISA of Small Molecules: A Proof of Concept Study.

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying target analytes in clinical and research laboratories. One of the main drawbacks of ELISA is the involvement of multiple washing steps that desorbs the capture antigen/antibody off the polystyrene plate, thereby producing inconsistent and erroneous data. To overcome the problem of desorption, we hypothesized that gelatin nanoparticles (GelNP) could serve as a "plate-adherent" substrate to irreversibly adhere the capture antigen/antibody of interest. We tested our hypothesis using GelNP-based substrate (Gel-BSA-OHG) to adhere 8-hydroxy-2'-deoxyguanosine (8-OHdG) to the polystyrene plate and assayed this molecule using the ELISA technique. The stability and ELISA performance of Gel-BSA-OHG was evaluated in comparison to the conventional substrate (BSA-OHG). Importantly, the Gel-BSA-OHG substrate was found to be more wash-resistant and consequently resulted in improved sensitivity, accuracy, and precision in the ELISA analysis of 8-OHdG. Finally, the scope of Gel-BSA-OHG substrate-based ELISA for clinical application was demonstrated by validating its ability to detect 8-OHdG in an artificial urine sample with high specificity.

Laboratory Tests for COVID-19: A Review of Peer-Reviewed Publications and Implications for Clinical Use.

Diagnostic tests for the coronavirus infection 2019 (COVID-19) are critical for prompt diagnosis, treatment and isolation to break the cycle of transmission. A positive real-time reverse-transcriptase polymerase chain reaction (RT-PCR), in conjunction with clinical and epidemiologic data, is the current standard for diagnosis, but several challenges still exist. Serological assays help to understand epidemiology better and to evaluate vaccine responses but they are unreliable for diagnosis in the acute phase of illness or assuming protective immunity. Serology is gaining attention, mainly because of convalescent plasma gaining importance as treatment for clinically worsening COVID-19 patients. We provide a narrative review of peer-reviewed research studies on RT-PCR, serology and antigen immune-assays for COVID-19, briefly describe their lab methods and discuss their limitations for clinical practice.

A novel crosslinker-free technique toward the fabrication of collagen microspheres.

Injectable collagen microspheres (CMs) have the potential to be an excellent tool to deliver various modulatory agents or to be used as a cellular transporter. A drawback has been the difficulty in producing reliable and spherical CMs. A crosslinker-free method to fabricate CMs was developed using liquid collagen (LC) in a water-in-oil emulsion process with varying concentrations of surfactant span-80. Different emulsion times of up to 16-hr were utilized to produce the CMs. Visual microscopy and scanning electron microscopy were utilized to determine the morphology of the CMs. To determine the fibril nature of the CMs, focus ion beam milling, energy dispersive spectroscopy, and Fourier Transformation-Infrared spectroscopy were performed. A cell biocompatibility study was performed to assess the biocompatibility of the CMs. The results demonstrated that consistent spherical CMs were achievable by changing the span-80 concentration. The CMs were fibrilized not only at the surface, but also at the core. Both the 1- and 16-hr emulsion time demonstrated biocompatibility and it appeared that the cells preferentially adhered to the CMs. This crosslinker-free method to fabricate CMs resulted in spherical, stable, biocompatible CMs, and could be an excellent technique for multiple tissue engineering applications.

Diabetic Retinopathy Screening Using a Gold Nanoparticle-Based Paper Strip Assay for the At-Home Detection of the Urinary Biomarker 8-Hydroxy-2'-Deoxyguanosine.

PURPOSE: We sought to assess a smartphone-based, gold nanoparticle-based colorimetric lateral flow immunoassay paper sensor for quantifying urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for diabetic retinopathy (DR) screening. METHODS: Paper strips incorporate gold nanoparticle-8-OHdG antibody conjugates that produce color changes that are proportional to urine 8-OHdG and that are discernible on a smartphone camera photograph. Paper strip accuracy, precision, and stability studies were performed with 8-OHdG solutions of varying concentrations. Urine was collected from 97 patients with diabetes who were receiving DR screening examinations, including 7-field fundus photographs. DR was graded by standard methods as either low risk (no or mild DR) or high risk (moderate or severe DR). Paper sensor assays were performed on urine samples from patients and 8-OHdG values were correlated with DR grades. The differences in 8-OHdG values between the low- and high-risk groups were analyzed for outliers to identify the threshold 8-OHdG value that would minimize false-negative results. RESULTS: Lateral flow immunoassay paper strips quantitatively measure 8-OHdG and were found to be accurate, precise, and stable. Average urine 8-OHdG concentrations in study patients were 22 ± 10 ng/mg of creatinine in the low-risk group and 55 ± 11 ng/mg of creatinine in the high-risk group. Screening cutoff values of 8-OHdG >50 ng/mg of creatinine or urine creatinine >1.5 mg minimized screen failures, with 91% sensitivity and 81% specificity. CONCLUSIONS: Urinary 8-OHdG is a useful biomarker to screen DR. Quantitative 8-OHdG detection with the lateral flow immunoassay paper sensor and smartphone camera demonstrates its potential in DR screening. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Silencing AXL by covalent siRNA-gelatin-antibody nanoconjugate inactivates mTOR/EMT pathway and stimulates p53 for TKI sensitization in NSCLC.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality with the 5-year survival rate at a dismal 16% for the past 40 years. Drug resistance is a major obstacle to achieving long-term patient survival. Identifying and validating molecular biomarkers responsible for resistance and thereby adopting multi-directional therapy is necessary to improve the survival rate. Previous studies indicated ~20% of tyrosine kinase inhibitor (TKI) resistant NSCLC patients overexpress AXL with increase in EMT and decrease in p53 expression. To overcome the resistance, we designed gelatin nanoparticles covalently conjugated with EGFR targeting antibody and siRNA (GAbsiAXL). GAbsiAXL efficiently silences AXL, decreases mTOR and EMT signaling with concomitant increase in p53 expression. Because of the molecular changes, the AXL silencing sensitizes the cells to TKI. Our results show AXL overexpression has an important role in driving TKI resistance through close association with energy-dependent mitochondrial pathways.

Correction to: Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

The authors would like to bring to the reader's attention that the Clarke error grid plot presented in Fig. 3 was generated using codes adapted from following reference.

Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

Optical monitoring of blood glucose levels for non-invasive diagnosis is a growing area of research. Recent efforts in this direction have been inclined towards reducing the requirement of calibration framework. Here, we are presenting a systematic investigation on the influence of variation in the ratio of calibration and validation points on the prospective predictive accuracy of spectral models. A fiber-optic probe coupled Raman system has been employed for transcutaneous measurements. Limit of agreement analysis between serum and partial least square regression predicted spectroscopic glucose values has been performed for accurate comparison. Findings are suggestive of strong predictive accuracy of spectroscopic models without requiring substantive calibration measurements. Graphical abstract.

Targeting HMGA protein inhibits retinoblastoma cell proliferation.

We describe a novel synthetic strategy for conjugating HMGA2 siRNA and the HMGA aptamer to the nucleolin aptamer and nucleolin antibody, respectively. Our studies demonstrate that these conjugates inhibit cell proliferation in retinoblastoma cells.

Identification and Validation of a PD-L1 Binding Peptide for Determination of PDL1 Expression in Tumors.

Blocking the interaction between Programmed Death Ligand 1 (PD-L1) and its receptor, PD-1, is an effective method of treating many types of cancers. Certain tumors overexpress PD-L1, causing host immune cells that express PD-1 to bind PD-L1 and cease killing the tumor. Inhibition of PD-L1 and PD-1 binding can restore host immunity towards tumor killing, and many new drugs have been developed to target this interaction. Current methods of PD-L1 diagnosis have shown to vary based on the antibody, detection kit brand, antigen retrieval method, and clinically defined methods by the FDA. To refine detection of PD-L1, we have identified a peptide, RK-10, and used it to detect PD-L1 expressing tumors with immunohistochemistry or flow cytometry. Flow cytometry was performed on cell lines and patient tissues using a fluorescent peptide (RK-10-Cy5). Immunohistochemistry using a biotin-modified peptide (RK-10-Biotin) was tested against the FDA-approved SP263 clone on biopsied patient tissues. For this study, we evaluated specificity of RK-10 using IHC in over 200 patient tissues, including NSCLC and Hodgkin's Lymphoma. RK-10 shows staining in the tumor regions of FFPE tissues where the SP263 kit does not. RK-10-Cy5 peptide also demonstrates PD-L1 detection in NSCLC, breast, squamous cell carcinoma, and melanoma.

Gut Dysbiosis and Neurobehavioral Alterations in Rats Exposed to Silver Nanoparticles.

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are being used in non-edible and edible consumer products. It is not clear though if exposure to these chemicals can exert toxic effects on the host and gut microbiome. Conflicting studies have been reported on whether AgNPs result in gut dysbiosis and other changes within the host. We sought to examine whether exposure of Sprague-Dawley male rats for two weeks to different shapes of AgNPs, cube (AgNC) and sphere (AgNS) affects gut microbiota, select behaviors, and induces histopathological changes in the gastrointestinal system and brain. In the elevated plus maze (EPM), AgNS-exposed rats showed greater number of entries into closed arms and center compared to controls and those exposed to AgNC. AgNS and AgNC treated groups had select reductions in gut microbiota relative to controls. Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus were decreased in AgNC exposed group, whereas, Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica were reduced in AgNS exposed group. Bacterial reductions correlated with select behavioral changes measured in the EPM. No significant histopathological changes were evident in the gastrointestinal system or brain. Findings suggest short-term exposure to AgNS or AgNC can lead to behavioral and gut microbiome changes.

In vitro/in vivo "peeling" of multilayered aminocarboxylate gold nanoparticles evidenced by a kinetically stable 99mTc-label.

A thiolated bombesin peptide was conjugated to Au-DTDTPA nanoconstructs to obtain BBN-Au-DTDTPA targeted to the gastrin releasing peptide receptor (GRPr). Different analytical techniques showed that this conjugate shares similar physico-chemical properties with Au-DTDTPA; HPLC and XPS analyses corroborated the attachment of the bioactive peptide to the AuNPs surface. Competitive binding assays in PC3 cancer cells showed that these BBN-containing AuNPs have high affinity for GRPr. BBN-Au-DTDTPA was successfully radiolabeled with 99mTc and showed high in vitro stability towards different biological media and substrates, except for glutathione (GSH). In vitro and in vivo studies, based on gamma-counting (99mTc content) and neutron activation analysis (Au content), indicated the release of the DTDTPA coating from the AuNPs. Probably, the "peeling" of the layered-aminocarboxylate coating is GSH-mediated and involves the cleavage of the DTDTPA disulfide bonds and/or Au-S bonds. These results render BBN-Au-DTDTPA an interesting platform deserving further evaluation in target-specific GSH-mediated drug delivery.

Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to KRAS mutant NSCLC dissociates GAB1-SHP2 post oncogene knockdown.

A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation.

DIETARY SILVER NANOPARTICLES REDUCE FITNESS IN A BENEFICIAL, BUT NOT PEST, INSECT SPECIES.

Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.

Three-Dimensional Nanocomposites: Fluidics Driven Assembly of Metal Nanoparticles on Protein Nanostructures and Their Cell-Line-Dependent Intracellular Trafficking Pattern.

Three-dimensional nanocomposites prepared using two different families of nanomaterials holds significant relevance pertaining to biological applications. However, integration of the two distinct nanomaterials with precision to control the overall compositional homogeneity of the resulting 3D nanocomposite is a synthetic challenge. Conventional reactions result in nanocomposites with heterogeneous composition and render useless. To address this challenge, we have developed a fluidics-mediated process for controlling the interaction of nanoparticles to yield a compositional uniform multidimensional nanoparticle; as an example, we demonstrated the integration of gold nanoparticles on gelatin nanoparticles. The composition of the nanocomposite is controlled by reacting predetermined number of gold nanoparticles to a known number of thiolated gelatin nanoparticles at any given time within a defined cross-sectional area. Using the fluidics process, we developed nanocomposites of different composition: [gelatin nanoparticles-(gold nanoparticles)x] where xaverage = 2, 12, or 25. The nanocomposites were further surface conjugated with organic molecules such as fluorescent dye or polyethylene glycol (PEG) molecules. To study the biological behavior of nanocomposite, we investigated the cellular internalization and trafficking characteristics of nanocomposites in two human cancer cell lines. The nanocomposites exhibited a three-stage cellular release mechanism that enables the translocation of gold nanoparticles within various cellular compartments. In summary, the three-dimensional nanocomposite serves as a novel platform for developing well-defined protein-metal nanocomposites for potential drug delivery, sensory, and molecular imaging applications.

Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-Functionalized Radiolabeled Gold Nanoparticles in Tumor Mice.

To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of (67)Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7-14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice indicated that the presence of BBN influences to some degree the biological profile of the nanoconstructs. For IV administration, the receptor-mediated pathway appears to be outweighed by the EPR effect. By contrast, in IP administration, it is reasoned that the GRPr-mediated mechanism plays a role in pancreas uptake.