Upconversion nanoparticle-assisted single-molecule assay for detecting circulating antigens of aggressive prostate cancer.

Sensitive and quantitative detection of molecular biomarkers is crucial for the early diagnosis of diseases like metabolic syndrome and cancer. Here we present a single-molecule sandwich immunoassay by imaging the number of single nanoparticles to diagnose aggressive prostate cancer. Our assay employed the photo-stable upconversion nanoparticles (UCNPs) as labels to detect the four types of circulating antigens in blood circulation, including glypican-1 (GPC-1), leptin, osteopontin (OPN), and vascular endothelial growth factor (VEGF), as their serum concentrations indicate aggressive prostate cancer. Under a wide-field microscope, a single UCNP doped with thousands of lanthanide ions can emit sufficiently bright anti-Stokes' luminescence to become quantitatively detectable. By counting every single streptavidin-functionalized UCNP which specifically labeled on each sandwich immune complex across multiple fields of views, we achieved the Limit of Detection (LOD) of 0.0123 ng/ml, 0.2711 ng/ml, 0.1238 ng/ml, and 0.0158 ng/ml for GPC-1, leptin, OPN and VEGF, respectively. The serum circulating level of GPC-1, leptin, OPN, and VEGF in a mixture of 10 healthy normal human serum was 25.17 ng/ml, 18.04 ng/ml, 11.34 ng/ml, and 1.55 ng/ml, which was within the assay dynamic detection range for each analyte. Moreover, a 20% increase of GPC-1 and OPN was observed by spiking the normal human serum with recombinant antigens to confirm the accuracy of the assay. We observed no cross-reactivity among the four biomarker analytes, which eliminates the false positives and enhances the detection accuracy. The developed single upconversion nanoparticle-assisted single-molecule assay suggests its potential in clinical usage for prostate cancer detection by monitoring tiny concentration differences in a panel of serum biomarkers.

Development of thermosensitive hydrogel wound dressing containing Acinetobacter baumannii phage against wound infections.

With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, respectively. Excellent storage stability of the IME-AB2 phage in a ~18 wt% Poloxamer 407 (P407) hydrogel solution was first demonstrated with negligible titer loss (~0.5 log) in 24 months at 4 °C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37 °C demonstrated >5 log10 CFU/ml reduction against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% respectively). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% reduction in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections.

High resolution photopolymer for 3D printing of personalised microneedle for transdermal delivery of anti-wrinkle small peptide.

Acetyl-hexapeptide 3 (AHP-3) has good efficacy and safety profile as an anti-wrinkle small peptide. However, its skin permeation is poor due to its hydrophilicity and large molecular weight. 3D printing of personalised microneedles (MN), that contour to the skin surface, offers an attractive alternative for delivery for AHP-3. However, commercially available photocurable resin for 3D printing are not suitable for fabrication of drug loaded delivery systems. In this study, two liquid monomers, namely, polyethylene glycol diacrylate (PEGDA) and vinyl pyrrolidone (VP), were investigated at various proportions, for critical parameters such as mechanical strength of final polymer, rate of polymerisation, rate of swelling of final polymer, 3D printing resolution and safety profile of final polymer. The optimal resin, based on the above parameters, was that of ratio 7 VP: 3 PEGDA in weight. Drug loading into the optimal resin demonstrated that AHP-3 remained stable throughout the fabrication process and there was no effect on the physical properties of final polymer. Using a 3D scanned face model, a personalised MN patch was designed using computer aided design (CAD) software and subsequently fabricated using a Digital Light Processing (DLP) 3D printer, with the optimal resin. In vitro characterisation of fabricated MN patch demonstrated the ability to penetrate human cadaver dermatomed skin and the MN remained intact after compression. The final polymer also had minimal cytotoxicity to human dermal fibroblast. Therefore, personalised MN patch fabricated using the photopolymer can potentially be a novel approach to augment transdermal delivery of AHP-3 for effective wrinkle management.

Dispersion stability and biocompatibility of four ligand-exchanged NaYF4: Yb, Er upconversion nanoparticles.

Surface modification to obtain high dispersion stability and biocompatibility is a key factor for bio-application of upconversion nanoparticles (UCNPs). A systematic study of UCNPs modified with four hydrophilic molecules separately, comparing their dispersion stability in biological buffers and cellular biocompatibility is reported here. The results show that carboxyl-functionalized UCNPs (modified by 3,4-dihydrocinnamic acid (DHCA) or poly(monoacryloxyethyl phosphate (MAEP)) with negative surface charge have superior even-distribution in biological buffers compared to amino-functionalized UCNPs (modified by (aminomethyl)phosphonic (AMPA) or (3-Aminopropyl)triethoxysilane (APTES)) with positive surface charge. Subsequent investigation of cellular interactions revealed high levels of non-targeted cellular uptake of the particles modified with either of the three small molecules (AMPA, APTES, DHCA) and high levels of cytotoxicity when used at high concentrations. The particles were seen to be trapped as particle-aggregates within the cellular cytoplasm, leading to reduced cell viability and cell proliferation, along with dysregulation of the cell cycle as assessed by DNA content measurements. The dramatically reduced proportion of cells in G1 phase and the slightly increased proportion in G2 phase indicates inhibition of M phase, and the appearance of sub-G1 phase reflects cell necrosis. In contrast, MAEP-modified UCNPs are bio-friendly with increased dispersion stability in biological buffers, are non-cytotoxic, with negligible levels of non-specific cellular uptake and no effect on the cell cycle at both low and high concentrations. MAEP-modified UCNPs were further functionalized with streptavidin for intracellular microtubule imaging, and showed clear cytoskeletal structures via their upconversion luminescence. STATEMENT OF SIGNIFICANCE: Upconversion nanoparticles (UCNP) are an exciting potential nanomaterial for bio-applications. Their anti-Stokes luminescence makes them especially attractive to be used as imaging probes and thermal therapeutic reagents. Surface modification is the key to achieving stable and compatible hydrophilic-UCNPs. However, the lack of criteria to assess molecular ligands used for ligand exchange of nanoparticles has hampered the development of surface modification, and further limits UCNP's bio-application. Herein, we report a systematic comparative study of modified-UCNPs with four distinct hydrophilic molecules, assessing each particles' colloidal stability in biological buffers and their cellular biocompatibility. The protocol established here can serve as a potential guide for the surface modification of UCNPs in bio-applications.

Plerixafor alone for the mobilization and transplantation of HLA-matched sibling donor hematopoietic stem cells.

Plerixafor, a direct antagonist of CXCR4/stromal-derived factor 1, can safely and rapidly mobilize allografts without the use of granulocyte colony-stimulating factor (G-CSF). We conducted a phase 2, multicenter, prospective study of plerixafor-mobilized HLA-identical sibling allografts for allogeneic hematopoietic cell transplantation in recipients with hematological malignancies. Donors (n = 64) were treated with subcutaneous plerixafor (240 µg/kg) and started leukapheresis (LP) 4 hours later. The primary objective was to determine the proportion of donors who were successfully mobilized: defined as collection of ≥2.0 × 106 CD34+ cells per kilogram recipient weight in ≤2 LP sessions. Recipients subsequently received reduced intensity (RIC; n = 33) or myeloablative (MAC; n = 30) conditioning. Sixty-three of 64 (98%) donors achieved the primary objective. The median CD34+ cell dose per kilogram recipient weight collected within 2 days was 4.7 (0.9-9.6). Plerixafor was well tolerated with only grade 1 or 2 drug-related adverse events noted. Bone pain was not observed. Plerixafor-mobilized grafts engrafted promptly. One-year progression-free and overall survivals were 53% (95% confidence interval [CI], 36% to 71%) and 63% (95% CI, 46% to 79%) for MAC and 64% (95% CI, 47% to 79%) and 70% (95% CI, 53% to 84%) for RIC recipients, respectively. Donor toxicity was reduced relative to G-CSF mobilized related donors. This is the first multicenter trial to demonstrate that, as an alternative to G-CSF, plerixafor rapidly and safely mobilizes sufficient numbers of CD34+ cells from matched sibling donors for HCT. Engraftment was prompt, and outcomes in recipients were encouraging. This trial was registered at clinicaltrials.gov as #NCT01696461.

Spray-Dried Particles of Nitric Oxide-Modified Glutathione for the Treatment of Chronic Lung Infection.

Antibiotic resistance in pathogenic bacteria has emerged as a big challenge to human and animal health and significant economy loss worldwide. Development of novel strategies to tackle antibiotic resistance is of the utmost priority. In this study, we combined glutathione (GSH), a master antioxidant in all mammalian cells, and nitric oxide, a proven biofilm-dispersing agent, to produce GSNO. The resazurin biofilm viability assay, crystal violet biofilm assay, and confocal microscopy techniques showed that GSNO disrupted biofilms of both P. aeruginosa PAO1 and multidrug resistant A. baumaunii (MRAB 015069) more efficiently than GSH alone. In addition, GSNO showed a higher reduction in biofilm viability and biomass when combined with antibiotics. This combination treatment also inhibited A. baumaunii (MRAB 015069) growth and facilitated human foreskin fibroblast (HFF-1) confluence and growth simultaneously. A potentially inhalable GSNO powder with reasonable aerosol performance and antibiofilm activity was produced by spray drying. This combination shows promise as a novel formulation for treating pulmonary bacterial infections.

Systematic investigation of functional ligands for colloidal stable upconversion nanoparticles.

Despite intense efforts on surface functionalization to generate hydrophilic upconversion nanoparticles (UCNPs), long-term colloidal stability in physiological buffers remains a major concern. Here we quantitatively investigate the competitive adsorption of phosphate, carboxylic acid and sulphonic acid onto the surface of UCNPs and study their binding strength to identify the best conjugation strategy. To achieve this, we designed and synthesized three di-block copolymers composed of poly(ethylene glycol) methyl ether acrylate and a polymer block bearing phosphate, carboxylic or sulphonic acid anchoring groups prepared by an advanced polymerization technique, Reversible Addition Fragmentation Chain Transfer (RAFT). Analytical tools provide the evidence that phosphate ligands completely replaced all the oleic acid capping molecules on the surface of the UCNPs compared with incomplete ligand exchange by carboxylic and sulphonic acid groups. Meanwhile, simulated quantitative adsorption energy measurements confirmed that among the three functional groups, the calculated adsorption strength for phosphate anchoring ligands is higher which is in good agreement with experimental results regarding the best colloidal stability, especially in phosphate buffer solution. This finding suggests that polymers with multiple anchoring negatively charged phosphate moieties provide excellent colloidal stability for lanthanide ion-doped luminescent nanoparticles for various potential applications.

Exonuclease III-Assisted Upconversion Resonance Energy Transfer in a Wash-Free Suspension DNA Assay.

Sensitivity is the key in optical detection of low-abundant analytes, such as circulating RNA or DNA. The enzyme Exonuclease III (Exo III) is a useful tool in this regard; its ability to recycle target DNA molecules results in markedly improved detection sensitivity. Lower limits of detection may be further achieved if the detection background of autofluorescence can be removed. Here we report an ultrasensitive and specific method to quantify trace amounts of DNA analytes in a wash-free suspension assay. In the presence of target DNA, the Exo III recycles the target DNA by selectively digesting the dye-tagged sequence-matched probe DNA strand only, so that the amount of free dye removed from the probe DNA is proportional to the number of target DNAs. Remaining intact probe DNAs are then bound onto upconversion nanoparticles (energy donor), which allows for upconversion luminescence resonance energy transfer (LRET) that can be used to quantify the difference between the free dye and tagged dye (energy acceptor). This scheme simply avoids both autofluorescence under infrared excitation and many tedious washing steps, as the free dye molecules are physically located away from the nanoparticle surface, and as such they remain "dark" in suspension. Compared to alternative approaches requiring enzyme-assisted amplification on the nanoparticle surface, introduction of probe DNAs onto nanoparticles only after DNA hybridization and signal amplification steps effectively avoids steric hindrance. Via this approach, we have achieved a detection limit of 15 pM in LRET assays of human immunodeficiency viral DNA.

Pair correlation microscopy reveals the role of nanoparticle shape in intracellular transport and site of drug release.

Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for example with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.

Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth.

Neuroblastoma is frequently diagnosed at advanced stage disease and treatment includes high dose chemotherapy and surgery. Despite the use of aggressive therapy survival rates are poor and children that survive their disease experience long term side effects from their treatment, highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects, however its mechanism of action is unknown. Here we report that Dextran-Catechin, a conjugated form of catechin that increases serum stability, is preferentially and markedly active against neuroblastoma cells having high levels of intracellular copper, without affecting non-malignant cells. Copper transporter 1 (CTR1) is the main transporter of copper in mammalian cells and it is upregulated in neuroblastoma. Functional studies showed that depletion of CTR1 expression reduced intracellular copper levels and led to a decrease in neuroblastoma cell sensitivity to Dextran-Catechin, implicating copper in the activity of this compound. Mechanistically, Dextran-Catechin was found to react with copper, inducing oxidative stress and decreasing glutathione levels, an intracellular antioxidant and regulator of copper homeostasis. In vivo, Dextran-Catechin significantly attenuated tumour growth in human xenograft and syngeneic models of neuroblastoma. Thus, Dextran-Catechin targets copper, inhibits tumour growth, and may be valuable in the treatment of aggressive neuroblastoma and other cancers dependent on copper for their growth.

A Rationally Optimized Nanoparticle System for the Delivery of RNA Interference Therapeutics into Pancreatic Tumors in Vivo.

Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition-fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (TUBB3/ßIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced ßIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors.

Late Acute and Chronic Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation.

Several distinct graft-versus-host disease (GVHD)-related syndromes have been defined by the National Institutes of Health Consensus Conference. We enrolled a prospective cohort of 911 hematopoietic cell transplantation (HCT) recipients at 13 centers between March 2011 and May 2014 to evaluate 4 GVHD syndromes: late acute GVHD (aGVHD), chronic GVHD (cGVHD), bronchiolitis obliterans syndrome, and cutaneous sclerosis. The median age at HCT was 53.7 years. The majority of patients received a peripheral blood stem cell transplant (81%) following nonmyeloablative or reduced-intensity conditioning (55%). Pediatric age group and use of bone marrow and umbilical cord blood grafts were underrepresented in our cohort (≤11%). The cumulative incidence of late aGVHD (late onset and recurrent) was 10% at a median of 5.5 months post-HCT, that of cGVHD was 47% at a median of 7.4 months, that of bronchiolitis obliterans was 3% at a median of 12.2 months, and that of cutaneous sclerosis was 8% at a median onset of 14.0 months. Late aGVHD and bronchiolitis obliterans had particularly high nonrelapse mortality of 23% and 32%, respectively, by 2 years after diagnosis. The probability of late aGVHD- and cGVHD-free, relapse-free survival was 38% at 1 year post-HCT and 26% at 2 years post-HCT. This multicenter prospective study confirms the high rate of late aGVHD and cGVHD syndromes and supports the need for continuous close monitoring and development of more effective GVHD treatment strategies to improve HCT success.

Co-delivery of nitric oxide and antibiotic using polymeric nanoparticles.

The rise of hospital-acquired infections, also known as nosocomial infections, is a growing concern in intensive healthcare, causing the death of hundreds of thousands of patients and costing billions of dollars worldwide every year. In addition, a decrease in the effectiveness of antibiotics caused by the emergence of drug resistance in pathogens living in biofilm communities poses a significant threat to our health system. The development of new therapeutic agents is urgently needed to overcome this challenge. We have developed new dual action polymeric nanoparticles capable of storing nitric oxide, which can provoke dispersal of biofilms into an antibiotic susceptible planktonic form, together with the aminoglycoside gentamicin, capable of killing the bacteria. The novelty of this work lies in the attachment of NO-releasing moiety to an existing clinically used drug, gentamicin. The nanoparticles were found to release both agents simultaneously and demonstrated synergistic effects, reducing the viability of Pseudomonas aeruginosa biofilm and planktonic cultures by more than 90% and 95%, respectively, while treatments with antibiotic or nitric oxide alone resulted in less than 20% decrease in biofilm viability.

Iron oxide nanoparticle-mediated hyperthermia stimulates dispersal in bacterial biofilms and enhances antibiotic efficacy.

The dispersal phase that completes the biofilm lifecycle is of particular interest for its potential to remove recalcitrant, antimicrobial tolerant biofilm infections. Here we found that temperature is a cue for biofilm dispersal and a rise by 5 °C or more can induce the detachment of Pseudomonas aeruginosa biofilms. Temperature upshifts were found to decrease biofilm biomass and increase the number of viable freely suspended cells. The dispersal response appeared to involve the secondary messenger cyclic di-GMP, which is central to a genetic network governing motile to sessile transitions in bacteria. Furthermore, we used poly((oligo(ethylene glycol) methyl ether acrylate)-block-poly(monoacryloxy ethyl phosphate)-stabilized iron oxide nanoparticles (POEGA-b-PMAEP@IONPs) to induce local hyperthermia in established biofilms upon exposure to a magnetic field. POEGA-b-PMAEP@IONPs were non-toxic to bacteria and when heated induced the detachment of biofilm cells. Finally, combined treatments of POEGA-b-PMAEP@IONPs and the antibiotic gentamicin reduced by 2-log the number of colony-forming units in both biofilm and planktonic phases after 20 min, which represent a 3.2- and 4.1-fold increase in the efficacy against planktonic and biofilm cells, respectively, compared to gentamicin alone. The use of iron oxide nanoparticles to disperse biofilms may find broad applications across a range of clinical and industrial settings.

The use of nanoparticles to deliver nitric oxide to hepatic stellate cells for treating liver fibrosis and portal hypertension.

Polymeric nanoparticles are designed to transport and deliver nitric oxide (NO) into hepatic stellate cells (HSCs) for the potential treatment of both liver fibrosis and portal hypertension. The nanoparticles, incorporating NO donor molecules (S-nitrosoglutathione compound), are designed for liver delivery, minimizing systemic delivery of NO. The nanoparticles are decorated with vitamin A to specifically target HSCs. We demonstrate, using in vitro and in vivo experiments, that the targeted nanoparticles are taken up specifically by rat primary HSCs and the human HSC cell line accumulating in the liver. When nanoparticles, coated with vitamin A, release NO in liver cells, we find inhibition of collagen I and α-smooth muscle actin (α-SMA), fibrogenic genes associated with activated HSCs expression in primary rat liver and human activated HSCs without any obvious cytotoxic effects. Finally, NO-releasing nanoparticles targeted with vitamin A not only attenuate endothelin-1 (ET-1) which elicites HSC contraction but also acutely alleviates haemodynamic disorders in bile duct-ligated-induced portal hypertension evidenced by decreasing portal pressure (≈20%) and unchanging mean arterial pressure. This study clearly shows, for the first time, the potential for HSC targeted nanoparticle delivery of NO as a treatment for liver diseases with proven efficacy for alleviating both liver fibrosis and portal hypertension.

Cyclosporine in combination with mycophenolate mofetil versus methotrexate for graft versus host disease prevention in myeloablative HLA-identical sibling donor allogeneic hematopoietic cell transplantation.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic cell transplantation (HCT) despite current prophylaxis. Methotrexate (MTX) with a calcineurin inhibitor (CNI) is the current standard, however, has several toxicities. Mycophenolate mofetil (MMF) is frequently used in reduced-intensity HCT, but data in myeloablative transplants is limited. We thus retrospectively identified 241 patients who underwent myeloablative HCT from an HLA-identical sibling donor; 174 patients received cyclosporine (CSA) + MMF and 67 received CSA+MTX. Patients receiving MMF + CSA had rapid neutrophil (median 11 vs. 19 days with MTX+CSA), and platelet recovery (median 19 vs. 25 days), lower incidence of severe mucositis by OMAS (19% vs. 53%), and shorter length of hospital stay (median 25 vs. 36 days) (P < 0.001 for all comparisons). There were no significant differences in incidence of grade 2-4 (MMF+CSA 37% vs. MTX+CSA 39%) or 3-4 acute GVHD (17% vs. 12%), chronic GVHD (46% vs. 56%), relapse (28% vs. 27%), non-relapse mortality (20% vs. 27%), or overall survival (47% vs. 44%) (P = NS for all). However, in multivariable analysis, the use of MMF+CSA was associated with an increased risk of severe grade 3-4 acute GVHD (HR 2.92, 95% CI 1.2-7.15, P = 0.019). There were no differences between the two regimens in multivariable analyses for other survival outcomes. This analysis demonstrates that the use of MMF in myeloablative sibling donor transplantation is well tolerated. However, there may be an increased risk of severe GVHD with MMF+CSA compared to MTX+CSA. Further studies evaluating optimal dosing strategies are needed.

Nanoparticles based on star polymers as theranostic vectors: endosomal-triggered drug release combined with MRI sensitivity.

Dual-functional star polymers (diameters 15 nm) are synthesized producing nanoparticles with excellent colloidal stability in both water and serum. The nanoparticles are built with aldehyde groups in the core and activated esters in the arms. The different reactivity of the two functional groups to sequentially react with different amino compounds is exploited; doxorubicin (DOX) and 1-(5-amino-3-aza-2-oxypentyl)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (DO3A-tBu-NH2 )-a chelating agent effective for the complexation of Gadolinium ions (Gd). The activated ester group is employed to attach the DO3A chelating agent, while the aldehyde groups are exploited for DOX conjugation, providing a controlled release mechanism for DOX in acidic environments. DOX/Gd-loaded nanoparticles are rapidly taken up by MCF-7 breast cancer cells, subsequently releasing DOX as demonstrated using in vitro fluorescence lifetime imaging microscopy (FLIM). Endosomal, DOX release is observed, using a phasor plot representation of the fluorescence lifetime data, showing an increase of native DOX with time. The MRI properties of the stars are assessed and the relaxivity of Gd loaded in stars is three times higher than conventional organic Gd/DO3A complexes. The DOX/Gd-conjugated nanoparticles yield a similar IC50 to native DOX for breast cancer cell lines, confirming that DOX integrity is conserved during nanoparticle attachment and release.