Evaluating the efficacy of Rose Bengal-PVA combinations within PCL/PLA implants for sustained cancer treatment.

The current investigation aims to address the limitations of conventional cancer therapy by developing an advanced, long-term drug delivery system using biocompatible Rose Bengal (RB)-loaded polyvinyl alcohol (PVA) matrices incorporated into 3D printed polycaprolactone (PCL) and polylactic acid (PLA) implants. The anticancer drug RB's high solubility and low lipophilicity require frequent and painful administration to the tumour site, limiting its clinical application. In this study, RB was encapsulated in a PVA (RB@PVA) matrix to overcome these challenges and achieve a localised and sustained drug release system within a biodegradable implant designed to be implanted near the tumour site. The RB@PVA matrix demonstrated an RB loading efficiency of 77.34 ± 1.53%, with complete RB release within 30 min. However, when integrated into implants, the system provided a sustained RB release of 75.84 ± 8.75% over 90 days. Cytotoxicity assays on PC-3 prostate cancer cells indicated an IC50 value of 1.19 µM for RB@PVA compared to 2.49 µM for free RB, effectively inhibiting cancer cell proliferation. This innovative drug delivery system, which incorporates a polymer matrix within an implantable device, represents a significant advancement in the sustained release of hydrosoluble drugs. It holds promise for reducing the frequency of drug administration, thereby improving patient compliance and translating experimental research into practical therapeutic applications.

3D-printed implants loaded with acriflavine for glioblastoma treatment.

Drug delivery routes play an essential role in determining the efficacy and safety of medications. This study focused on the development and optimization of 3D-printed reservoir type implants as a combinational therapy drug delivery system for Glioblastoma Multiforme (GBM) post-surgery, possessing also antibacterial properties. In this study, we used a multimodal agent, Acriflavine (ACF) as an alternative drug to treat GBM. To date, ACF is used only as an antiseptic agent, although it has been shown to possess strong anticancer activities. ACF and a low molecular weight PCL were loaded into 3D-printed reservoir-type implants for sustained drug delivery. The study demonstrated that ACF implants exhibited sustained drug release kinetics, with faster release during the initial 30 days, followed by a gradual decrease over 90 days. This controlled release profile enhances the effectiveness of ACF delivery to tumour targets while minimizing side effects associated with systemic administration. In vitro experiments confirmed the inhibitory activity of ACF against GBM cells compared to non-tumour cells. The study also highlighted the bacteriostatic effects of ACF, making the implants potentially useful for post-surgery infection management, particularly against S. aureus, a common bacterial infection associated with brain surgery. The long-term drug-release capabilities of the implants make them attractive candidates for both tumour inhibition and antibacterial treatment. The study suggests that the developed ACF delivery systems have the potential for future clinical studies. Their ability to provide increased drug efficacy without systemic toxicity makes them promising candidates for cancer therapy and post-surgery infection management.

Immunotherapy for HPV negative head and neck squamous cell carcinoma.

Head and neck cancer (HNSCC) is the 8th most common cancer in the UK, with incidence increasing due to lifestyle factors such as tobacco and alcohol abuse. HNSCC is an immune-suppressive disease characterised by impaired cytokine secretion and dysregulation of immune infiltrate. As such, immunotherapy is a potential treatment option, with therapeutic cancer vaccination demonstrating the greatest potential. The success of cancer vaccination is dependent on informed antigen selection: an ideal antigen must be either tumour-specific or tumour-associated, as well as highly immunogenic. Stratification of the patient population for antigen expression and validated biomarkers are also vital. This review focuses on the latest developments in immunotherapy, specifically the development of therapeutic vaccines, and highlights successes, potential drawbacks and areas for future development. Immunotherapy approaches considered for HNSCC include monoclonal antibodies (mAb), Oncolytic viral (OV) therapies, Immune Checkpoint Inhibitors (ICIs) and cancer vaccines.

Microfluidic technologies in tumour metabolism.

The tumour microenvironment presents many challenges in the development and evaluation of new anti-cancer therapeutics. Enhanced understanding of the unique metabolic characteristics of cancer cells could provide valuable knowledge to develop broad-spectrum anti-neoplastic agents with reduced systemic toxicity. However, a major limitation is the lack of physiological relevance of many conventional in vitro models. Emerging microfluidic platform technologies, hold the potential of recapitulating the physiological and pathological features of the tumour microenvironment, thus increasing the relevance of pre-clinical experimental data. With precise manipulation of physical and chemical properties, the microfluidic based, tumour-on-chip authentically replicates the tumour growth environment, potentially helping accelerate the pre-clinical screening of novel anti-tumour drug combinations. Additionally, nanoscale vehicles produced using microfluidic technologies, offer an emerging solution to modulate the hydrophilicity and release kinetics of drugs that in the free state exhibit challenging pharmacokinetics. This article will review the current state-of-the-art in this space and outline important challenges that are yet to be overcome.

Formulating RALA/Au nanocomplexes to enhance nanoparticle internalisation efficiency, sensitising prostate tumour models to radiation treatment.

BACKGROUND: Gold nanoparticles (AuNP) are effective radiosensitisers, however, successful clinical translation has been impeded by short systemic circulation times and poor internalisation efficiency. This work examines the potential of RALA, a short amphipathic peptide, to enhance the uptake efficiency of negatively charged AuNPs in tumour cells, detailing the subsequent impact of AuNP internalisation on tumour cell radiation sensitivity. RESULTS: RALA/Au nanoparticles were formed by optimising the ratio of RALA to citrate capped AuNPs, with assembly occurring through electrostatic interactions. Physical nanoparticle characteristics were determined by UV-vis spectroscopy and dynamic light scattering. Nano-complexes successfully formed at w:w ratios > 20:1 (20 µg RALA:1 µg AuNP) yielding positively charged nanoparticles, sized < 110 nm with PDI values < 0.52. ICP-MS demonstrated that RALA enhanced AuNP internalisation by more than threefold in both PC-3 and DU145 prostate cancer cell models, without causing significant toxicity. Importantly, all RALA-AuNP formulations significantly increased prostate cancer cell radiosensitivity. This effect was greatest using the 25:1 RALA-AuNP formulation, producing a dose enhancement effect (DEF) of 1.54 in PC3 cells. Using clinical radiation energies (6 MV) RALA-AuNP also significantly augmented radiation sensitivity. Mechanistic studies support RALA-AuNP nuclear accumulation resulting in increased DNA damage yields. CONCLUSIONS: This is the first study to demonstrate meaningful radiosensitisation using low microgram AuNP treatment concentrations. This effect was achieved using RALA, providing functional evidence to support our previous imaging study indicating RALA-AuNP nuclear accumulation.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

Radiation Response in the Tumour Microenvironment: Predictive Biomarkers and Future Perspectives.

Radiotherapy (RT) is a primary treatment modality for a number of cancers, offering potentially curative outcomes. Despite its success, tumour cells can become resistant to RT, leading to disease recurrence. Components of the tumour microenvironment (TME) likely play an integral role in managing RT success or failure including infiltrating immune cells, the tumour vasculature and stroma. Furthermore, genomic profiling of the TME could identify predictive biomarkers or gene signatures indicative of RT response. In this review, we will discuss proposed mechanisms of radioresistance within the TME, biomarkers that may predict RT outcomes, and future perspectives on radiation treatment in the era of personalised medicine.

Clinical and functional characterization of CXCR1/CXCR2 biology in the relapse and radiotherapy resistance of primary PTEN-deficient prostate carcinoma.

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTEN LOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.

Exploiting Current Understanding of Hypoxia Mediated Tumour Progression for Nanotherapeutic Development.

Hypoxia is one of the most common phenotypes of malignant tumours. Hypoxia leads to the increased activity of hypoxia-inducible factors (HIFs), which regulate the expression of genes controlling a raft of pro-tumour phenotypes. These include maintenance of the cancer stem cell compartment, epithelial-mesenchymal transition (EMT), angiogenesis, immunosuppression, and metabolic reprogramming. Hypoxia can also contribute to the tumour progression in a HIF-independent manner via the activation of a complex signalling network pathway, including JAK-STAT, RhoA/ROCK, NF-κB and PI3/AKT. Recent studies suggest that nanotherapeutics offer a unique opportunity to target the hypoxic microenvironment, enhancing the therapeutic window of conventional therapeutics. In this review, we summarise recent advances in understanding the impact of hypoxia on tumour progression, while outlining possible nanotherapeutic approaches for overcoming hypoxia-mediated resistance.

Cell-Penetrating Peptides as a Tool for the Cellular Uptake of a Genetically Modified Nitroreductase for use in Directed Enzyme Prodrug Therapy.

Directed enzyme prodrug therapy (DEPT) involves the delivery of a prodrug-activating enzyme to a solid tumour site, followed by the subsequent activation of an administered prodrug. One of the most studied enzyme-prodrug combinations is the nitroreductase from Escherichia coli (NfnB) with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitro-benzamide]. One of the major issues faced by DEPT is the ability to successfully internalize the enzyme into the target cells. NfnB has previously been genetically modified to contain cysteine residues (NfnB-Cys) which bind to gold nanoparticles for a novel DEPT therapy called magnetic nanoparticle directed enzyme prodrug therapy (MNDEPT). One cellular internalisation method is the use of cell-penetrating peptides (CPPs), which aid cellular internalization of cargo. Here the cell-penetrating peptides: HR9 and Pep-1 were tested for their ability to conjugate with NfnB-Cys. The conjugates were further tested for their potential use in MNDEPT, as well as conjugating with the delivery vector intended for use in MNDEPT and tested for the vectors capability to penetrate into cells.

DNA vaccination via RALA nanoparticles in a microneedle delivery system induces a potent immune response against the endogenous prostate cancer stem cell antigen.

Castrate resistant prostate cancer (CRPC) remains a major challenge for healthcare professionals. Immunotherapeutic approaches, including DNA vaccination, hold the potential to harness the host's own immune system to mount a cell-mediated, anti-tumour response, capable of clearing disseminated tumour deposits. These anti-cancer vaccines represent a promising strategy for patients with advanced disease, however, to date DNA vaccines have demonstrated limited efficacy in clinical trials, owing to the lack of a suitable DNA delivery system. This study was designed to evaluate the efficacy of a two-tier delivery system incorporating cationic RALA/pDNA nanoparticles (NPs) into a dissolvable microneedle (MN) patch for the purposes of DNA vaccination against prostate cancer. Application of NP-loaded MN patches successfully resulted in endogenous production of the encoded Prostate Stem Cell Antigen (PSCA). Furthermore, immunisation with RALA/pPSCA loaded MNs elicited a tumour-specific immune response against TRAMP-C1 tumours ex vivo. Finally, vaccination with RALA/pPSCA loaded MNs demonstrated anti-tumour activity in both prophylactic and therapeutic prostate cancer models in vivo. This is further evidence that this two-tier MN delivery system is a robust platform for prostate cancer DNA vaccination. STATEMENT OF SIGNIFICANCE: This research describes the development and utilisation of our unique microneedle (MN) DNA delivery system, which enables penetration through the stratum corneum and deposition of the DNA within the highly immunogenic skin layers via a dissolvable MN matrix, and facilitates cellular uptake via complexation of pDNA cargo into nanoparticles (NPs) with the RALA delivery peptide. We report for the first time on using the NP-MN platform to immunise mice with encoded Prostate Stem Cell Antigen (mPSCA) for prostate cancer DNA vaccination. Application of the NP-MN system resulted in local mPSCA expression in vivo. Furthermore, immunisation with the NP-MN system induced a tumour-specific cellular immune response, and inhibited the growth of TRAMP-C1 prostate tumours in both prophylactic and therapeutic challenge models in vivo.

Towards photon radiotherapy treatment planning with high Z nanoparticle radiosensitisation agents: the Relative Biological Effective Dose (RBED) framework.

A novel treatment planning framework, the Relative Biological Effective Dose (RBED), for high Z nanoparticle (NP)-enhanced photon radiotherapy is developed and tested in silico for the medical exemplar of neoadjuvant (preoperative) breast cancer MV photon radiotherapy. Two different treatment scenarios, conventional and high Z NP enhanced, were explored with a custom Geant4 application that was developed to emulate the administration of a single 2 Gy fraction as part of a 50 Gy radiotherapy treatment plan. It was illustrated that there was less than a 1% difference in the dose deposition throughout the standard and high Z NP-doped adult female phantom. Application of the RBED framework found that the extent of possible biological response with high Z NP doping was great than expected via the dose deposition alone. It is anticipated that this framework will assist the scientific community in future high Z NP-enhanced in-silico, pre-clinical and clinical trials.

Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer.

BACKGROUND: Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from the presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study examines the use of RALA to deliver a plasmid encoding inducible nitric oxide synthase (iNOS) as an anti-cancer treatment. METHODS: The physiochemical properties of the RALA/DNA nanoparticles were characterized via dynamic light scattering and transmission electron microscopy. The nanoparticles were labelled with fluorophores and tracked over time using confocal microscopy with orthogonal sections to determine cellular location. In vitro studies were employed to determine functionality of the nanoparticles both for pEGFP-N1 and CMV-iNOS. Nanoparticles were injected intravenously into C57/BL6 mice with blood and serum samples analysed for immune response. PC3-luc2M cells were injected into the left ventricle of SCID mice followed by treatment with RALA/CMV-iNOS nanoparticles to evaluate the tumour response in a metastatic model of prostate cancer. RESULTS: Functional cationic nanoparticles were produced with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles into immunocompetent mice did not produce any immunological response: neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically delivered RALA/CMV-iNOS significantly improved the survival of mice. CONCLUSION: These studies further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer.

Unraveling the cell-type dependent radiosensitizing effects of gold through the development of a multifunctional gold nanoparticle.

The radiosensitizing efficacy of gold is well established, however, there remain several significant barriers to the successful clinical translation of nano-sized gold particles (AuNPs). These barriers include: retaining stability in relevant biological sera, demonstrating effectiveness at clinically relevant AuNP concentrations and identifying the biological context where significant benefit is most likely to be achieved. Herein we have developed a AuNP preparation, stress-tested to provide effective protection from salt and serum mediated agglomeration. Furthermore, the core AuNP is co-functionalized with two biologically derived peptides designed to enhance endocytosis and promote endosomal escape, thus maximizing intracellular AuNP surface area. In summary, these investigations demonstrate restored AuNP internalization using the co-functionalized preparation that generated significant radiosensitization, in both in vitro and in vivo models, at clinically viable treatment concentrations. Furthermore, we have identified an underpinning biological mechanism in the inherent radical scavenging capacity that could be used to predict radiosensitizing efficacy.

Enhanced nanoparticle delivery exploiting tumour-responsive formulations.

Nanoparticles can be used as drug carriers, contrast agents and radiosensitisers for the treatment of cancer. Nanoparticles can either passively accumulate within tumour sites, or be conjugated with targeting ligands to actively enable tumour deposition. With respect to passive accumulation, particles < 150 nm accumulate with higher efficiency within the tumour microenvironment, a consequence of the enhanced permeability and retention effect. Despite these favourable properties, clinical translation of nano-therapeutics is inhibited due to poor in vivo stability, biodistribution and target cell internalisation. Nano-therapeutics can be modified to exploit features of the tumour microenvironment such as elevated hypoxia, increased pH and a compromised extracellular matrix. This is in contrast to cytotoxic chemotherapies which generally do not exploit the characteristic pathological features of the tumour microenvironment, and as such are prone to debilitating systemic toxicities. This review examines strategies for tumour microenvironment targeting to improve nanoparticle delivery, with particular focus on the delivery of nucleic acids and gold nanoparticles. Evidence for key research areas and future technologies are presented and critically evaluated. Among the most promising technologies are the development of next-generation cell penetrating peptides and the incorporation of micro-environment responsive stealth molecules.

A hierarchical Bayesian approach to calibrating the linear-quadratic model from clonogenic survival assay data.

We propose a Bayesian hierarchical model applicable to the calibration of the linear-quadratic model of radiation dose-response. Experimental data used in model calibration were taken from a clonogenic survival assay conducted on human breast cancer cells (MDA-MB-231) across a range of radiation doses (0-6Gy). Employing Markov-chain Monte Carlo methods, we calibrated the proposed Bayesian hierarchical model, computed posterior distributions for the model parameters and survival fraction dose-response probability densities. Key contributions include the proposal of a model that incorporates multiple sources of inter- and intra-experiment variability commonly neglected in the standard frequentist approach and its subsequent application to in vitro experimental data.

Erythropoietin drives breast cancer progression by activation of its receptor EPOR.

Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood.Here, we have examined the functional role of the EPO-EPOR axis in pre-clinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth.Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.

Systemic RALA/iNOS Nanoparticles: A Potent Gene Therapy for Metastatic Breast Cancer Coupled as a Biomarker of Treatment.

This study aimed to determine the therapeutic benefit of a nanoparticular formulation for the delivery of inducible nitric oxide synthase (iNOS) gene therapy in a model of breast cancer metastasis. Nanoparticles comprising a cationic peptide vector, RALA, and plasmid DNA were formulated and characterized using a range of physiochemical analyses. Nanoparticles complexed using iNOS plasmids and RALA approximated 60 nm in diameter with a charge of 25 mV. A vector neutralization assay, performed to determine the immunogenicity of nanoparticles in immunocompetent C57BL/6 mice, revealed that no vector neutralization was evident. Nanoparticles harboring iNOS plasmids (constitutively active cytomegalovirus [CMV]-driven or transcriptionally regulated human osteocalcin [hOC]-driven) evoked iNOS protein expression and nitrite accumulation and impaired clonogenicity in the highly aggressive MDA-MB-231 human breast cancer model. Micrometastases of MDA-MB-231-luc-D3H1 cells were established in female BALB/c SCID mice by intracardiac delivery. Nanoparticulate RALA/CMV-iNOS or RALA/hOC-iNOS increased median survival in mice bearing micrometastases by 27% compared with controls and also provoked elevated blood nitrite levels. Additionally, iNOS gene therapy sensitized MDA-MB-231-luc-D3H1 tumors to docetaxel treatment. Studies demonstrated that systemically delivered RALA-iNOS nanoparticles have therapeutic potential for the treatment of metastatic breast cancer. Furthermore, detection of nitrite levels in the blood serves as a reliable biomarker of treatment.

Preclinical evaluation of gold-DTDTPA nanoparticles as theranostic agents in prostate cancer radiotherapy.

AIM: Gold nanoparticles have attracted significant interest in cancer diagnosis and treatment. Herein, we evaluated the theranostic potential of dithiolated diethylenetriamine pentaacetic acid (DTDTPA) conjugated AuNPs (Au@DTDTPA) for CT-contrast enhancement and radiosensitization in prostate cancer. MATERIALS & METHODS: In vitro assays determined Au@DTDTPA uptake, cytotoxicity, radiosensitizing potential and DNA damage profiles. Human PC3 xenograft tumor models were used to determine CT enhancement and radiation modulating effects in vivo. RESULTS: Cells exposed to nanoparticles and radiation observed significant additional reduction in survival compared with radiation only. Au@DTDTPA produced a CT enhancement of 10% and a significant extension in tumor growth delay from 16.9 days to 38.3 compared with radiation only. CONCLUSION: This study demonstrates the potential of Au@DTDTPA to enhance CT-image contrast and simultaneously increases the radiosensitivity of prostate tumors.

A comparison of gold nanoparticle surface co-functionalization approaches using Polyethylene Glycol (PEG) and the effect on stability, non-specific protein adsorption and internalization.

To create clinically useful gold nanoparticle (AuNP) based cancer therapeutics it is necessary to co-functionalize the AuNP surface with a range of moieties; e.g. Polyethylene Glycol (PEG), peptides and drugs. AuNPs can be functionalized by creating either a mixed monolayer by attaching all the moieties directly to the surface using thiol chemistry, or by binding groups to the surface by means of a bifunctional polyethylene glycol (PEG) linker. The linker methodology has the potential to enhance bioavailability and the amount of functional agent that can be attached. While there is a large body of published work using both surface arrangements independently, the impact of attachment methodology on stability, non-specific protein adsorption and cellular uptake is not well understood, with no published studies directly comparing the two most frequently employed approaches. This paper compares the two methodologies by synthesizing and characterizing PEG and Receptor Mediated Endocytosis (RME) peptide co-functionalized AuNPs prepared using both the mixed monolayer and linker approaches. Successful attachment of both PEG and RME peptide using the two methods was confirmed using Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy and gel electrophoresis. It was observed that while the 'as synthesized' citrate capped AuNPs agglomerated under physiological salt conditions, all the mixed monolayer and PEG linker capped samples remained stable at 1M NaCl, and were stable in PBS over extended periods. While it was noted that both functionalization methods inhibited non-specific protein attachment, the mixed monolayer samples did show some changes in gel electrophoresis migration profile after incubation with fetal calf serum. PEG renders the AuNP stable in-vivo however, studies with MDA-MB-231 and MCF 10A cell lines indicated that functionalization with PEG, blocks cellular uptake. It was observed that co-functionalization with RME peptide using both the mixed monolayer and PEG linker methods greatly enhanced cellular internalization compared to PEG capped AuNPs.