I-SPY COVID adaptive platform trial for COVID-19 acute respiratory failure: rationale, design and operations.

INTRODUCTION: The COVID-19 pandemic brought an urgent need to discover novel effective therapeutics for patients hospitalised with severe COVID-19. The Investigation of Serial studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis (ISPY COVID-19 trial) was designed and implemented in early 2020 to evaluate investigational agents rapidly and simultaneously on a phase 2 adaptive platform. This manuscript outlines the design, rationale, implementation and challenges of the ISPY COVID-19 trial during the first phase of trial activity from April 2020 until December 2021. METHODS AND ANALYSIS: The ISPY COVID-19 Trial is a multicentre open-label phase 2 platform trial in the USA designed to evaluate therapeutics that may have a large effect on improving outcomes from severe COVID-19. The ISPY COVID-19 Trial network includes academic and community hospitals with significant geographical diversity across the country. Enrolled patients are randomised to receive one of up to four investigational agents or a control and are evaluated for a family of two primary outcomes-time to recovery and mortality. The statistical design uses a Bayesian model with 'stopping' and 'graduation' criteria designed to efficiently discard ineffective therapies and graduate promising agents for definitive efficacy trials. Each investigational agent arm enrols to a maximum of 125 patients per arm and is compared with concurrent controls. As of December 2021, 11 investigational agent arms had been activated, and 8 arms were complete. Enrolment and adaptation of the trial design are ongoing. ETHICS AND DISSEMINATION: ISPY COVID-19 operates under a central institutional review board via Wake Forest School of Medicine IRB00066805. Data generated from this trial will be reported in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: NCT04488081.

Oxaliplatin-DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy.

FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 µmol/L) or diagnostic microdose (1 µmol/L) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [14C]oxaliplatin prior to treatment, as well as a second [14C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

Radiocarbon Tracers in Toxicology and Medicine: Recent Advances in Technology and Science.

This review summarizes recent developments in radiocarbon tracer technology and applications. Technologies covered include accelerator mass spectrometry (AMS), including conversion of samples to graphite, and rapid combustion to carbon dioxide to enable direct liquid sample analysis, coupling to HPLC for real-time AMS analysis, and combined molecular mass spectrometry and AMS for analyte identification and quantitation. Laser-based alternatives, such as cavity ring down spectrometry, are emerging to enable lower cost, higher throughput measurements of biological samples. Applications covered include radiocarbon dating, use of environmental atomic bomb pulse radiocarbon content for cell and protein age determination and turnover studies, and carbon source identification. Low dose toxicology applications reviewed include studies of naphthalene-DNA adduct formation, benzo[a]pyrene pharmacokinetics in humans, and triclocarban exposure and risk assessment. Cancer-related studies covered include the use of radiocarbon-labeled cells for better defining mechanisms of metastasis and the use of drug-DNA adducts as predictive biomarkers of response to chemotherapy.

Correlation of Platinum Cytotoxicity to Drug-DNA Adduct Levels in a Breast Cancer Cell Line Panel.

Platinum drugs, including carboplatin and oxaliplatin, are commonly used chemotherapy drugs that kill cancer cells by forming toxic drug-DNA adducts. These drugs have a proven, but modest, efficacy against several aggressive subtypes of breast cancer but also cause several side effects that can lead to the cessation of treatment. There is a clinical need to identify patients who will respond to platinum drugs in order to better inform clinical decision making. Diagnostic microdosing involves dosing patients or patient samples with subtherapeutic doses of radiolabeled platinum followed by measurement of platinum-DNA adducts in blood or tumor tissue and may be used to predict patient response. We exposed a panel of six breast cancer cell lines to 14C-labeled carboplatin or oxaliplatin at therapeutic and microdose (1% therapeutic dose) concentrations for a range of exposure lengths and isolated DNA from the cells. The DNA was converted to graphite, and measurement of radiocarbon due to platinum-DNA adduction was quantified via accelerator mass spectrometry (AMS). We observed a linear correlation in adduct levels between the microdose and therapeutic dose, and the level of platinum-DNA adducts corresponded to cell line drug sensitivity for both carboplatin and oxaliplatin. These results showed a clear separation in adduct levels between the sensitive and resistant groups of cell lines that could not be fully explained or predicted by changes in DNA repair rates or mutations in DNA repair genes. Further, we were able to quantitate oxaliplatin-DNA adducts in the blood and tumor tissue of a metastatic breast cancer patient. Together, these data support the use of diagnostic microdosing for predicting patient sensitivity to platinum. Future studies will be aimed at replicating this data in a clinical feasibility trial.

Toward Predicting Acute Myeloid Leukemia Patient Response to 7 + 3 Induction Chemotherapy via Diagnostic Microdosing.

Acute myeloid leukemia (AML) is a rare yet deadly cancer of the blood and bone marrow. Presently, induction chemotherapy with the DNA damaging drugs cytarabine (ARA-C) and idarubicin (IDA), known as 7 + 3, is the standard of care for most AML patients. However, 7 + 3 is a relatively ineffective therapy, particularly in older patients, and has serious therapy-related toxicities. Therefore, a diagnostic test to predict which patients will respond to 7 + 3 is a critical unmet medical need. We hypothesize that a threshold level of therapy-induced 7 + 3 drug-DNA adducts determines cytotoxicity and clinical response. We further hypothesize that in vitro exposure of AML cells to nontoxic diagnostic microdoses enables prediction of the ability of AML cells to achieve that threshold during treatment. Our test involves dosing cells with very low levels of 14C-labeled drug followed by DNA isolation and quantification of drug-DNA adducts via accelerator mass spectrometry. Here, we have shown proof of principle by correlating ARA-C- and DOX-DNA adduct levels with cellular IC50 values of paired sensitive and resistant cancer cell lines and AML cell lines. Moreover, we have completed a pilot retrospective trial of diagnostic microdosing for 10 viably cryopreserved primary AML samples and observed higher ARA-C- and DOX-DNA adducts in the 7 + 3 responders than nonresponders. These initial results suggest that diagnostic microdosing may be a feasible and useful test for predicting patient response to 7 + 3 induction chemotherapy, leading to improved outcomes for AML patients and reduced treatment-related morbidity and mortality.

Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications.

Nanolipoprotein particles (NLPs) are reconstituted high-density lipoproteins, consisting of a phospholipid bilayer stabilized by an apolipoprotein scaffold protein. This class of nanoparticle has been a vital tool in the study of membrane proteins, and in recent years has been increasingly used for in vivo applications. Previous work demonstrated that the composition of the lipid bilayer component affects the stability of these particles in serum solutions. In the current study, NLPs assembled with phosphatidylcholine lipids featuring different acyl chain structures were systematically tested to understand the effect that lipid composition has on NLP stability in both neat serum and cell culture media supplemented with 10% serum by volume. The time at which 50% of the particles dissociate, as well as the fraction of the initial population that remains resistant to dissociation, were correlated to key parameters obtained from all-atom simulations of the corresponding lipid bilayers. A significant correlation was observed between the compressibility modulus of the lipid bilayer and particle stability in these complex biological milieu. These results can be used as a reference to tune the stability of these versatile biological nanoparticles for in vitro and in vivo applications.

COX-2/sEH Dual Inhibitor PTUPB Potentiates the Antitumor Efficacy of Cisplatin.

Cisplatin-based therapy is highly toxic, but moderately effective in most cancers. Concurrent inhibition of cyclooxygenase-2 (COX-2) and soluble epoxide hydrolase (sEH) results in antitumor activity and has organ-protective effects. The goal of this study was to determine the antitumor activity of PTUPB, an orally bioavailable COX-2/sEH dual inhibitor, in combination with cisplatin and gemcitabine (GC) therapy. NSG mice bearing bladder cancer patient-derived xenografts were treated with vehicle, PTUPB, cisplatin, GC, or combinations thereof. Mouse experiments were performed with two different PDX models. PTUPB potentiated cisplatin and GC therapy, resulting in significantly reduced tumor growth and prolonged survival. PTUPB plus cisplatin was no more toxic than cisplatin single-agent treatment as assessed by body weight, histochemical staining of major organs, blood counts, and chemistry. The combination of PTUPB and cisplatin increased apoptosis and decreased phosphorylation in the MAPK/ERK and PI3K/AKT/mTOR pathways compared with controls. PTUPB treatment did not alter platinum-DNA adduct levels, which is the most critical step in platinum-induced cell death. The in vitro study using the combination index method showed modest synergy between PTUPB and platinum agents only in 5637 cell line among several cell lines examined. However, PTUPB is very active in vivo by inhibiting angiogenesis. In conclusion, PTUPB potentiated the antitumor activity of cisplatin-based treatment without increasing toxicity in vivo and has potential for further development as a combination chemotherapy partner. Mol Cancer Ther; 17(2); 474-83. ©2017 AACR.

The Phosphatidylinositol 3-Kinase Pathway as a Potential Therapeutic Target in Bladder Cancer.

Purpose: Activation of the PI3K pathway occurs in over 40% of bladder urothelial cancers. The aim of this study is to determine the therapeutic potential, the underlying action, and the resistance mechanisms of drugs targeting the PI3K pathway.Experimental Design: Urothelial cancer cell lines and patient-derived xenografts (PDXs) were analyzed for alterations of the PI3K pathway and for their sensitivity to the small-molecule inhibitor pictilisib alone and in combination with cisplatin and/or gemcitabine. Potential predictive biomarkers for pictilisib were evaluated, and RNA sequencing was performed to explore drug resistance mechanisms.Results: The bladder cancer cell line TCCSUP, which harbors a PIK3CA E545K mutation, was sensitive to pictilisib compared to cell lines with wild-type PIK3CA Pictilisib exhibited stronger antitumor activity in bladder cancer PDX models with PI3KCA H1047R mutation or amplification than the control PDX model. Pictilisib synergized with cisplatin and/or gemcitabine in vitro, significantly delayed tumor growth, and prolonged survival compared with single-drug treatment in the PDX models. The phosphorylation of ribosomal protein S6 correlated with response to pictilisib both in vitro and in vivo, and could potentially serve as a biomarker to predict response to pictilisib. Pictilisib activated the compensatory MEK/ERK pathway that likely contributed to pictilisib resistance, which was reversed by cotreatment with the RAF inhibitor sorafenib. RNA sequencing of tumors resistant to treatment suggested that LSP1 downregulation correlated with drug resistance.Conclusions: These preclinical results provide new insights into the therapeutic potential of targeting the PI3K pathway for the treatment of bladder cancer. Clin Cancer Res; 23(21); 6580-91. ©2017 AACR.

Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system.

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

A diagnostic microdosing approach to investigate platinum sensitivity in non-small cell lung cancer.

The platinum-based drugs cisplatin, carboplatin and oxaliplatin are often used for chemotherapy, but drug resistance is common. The prediction of resistance to these drugs via genomics is a challenging problem since hundreds of genes are involved. A possible alternative is to use mass spectrometry to determine the propensity for cells to form drug-DNA adducts-the pharmacodynamic drug-target complex for this class of drugs. The feasibility of predictive diagnostic microdosing was assessed in non-small cell lung cancer (NSCLC) cell culture and a pilot clinical trial. Accelerator mass spectrometry (AMS) was used to quantify [14 C]carboplatin-DNA monoadduct levels in the cell lines induced by microdoses and therapeutic doses of carboplatin, followed by correlation with carboplatin IC50 values for each cell line. The adduct levels in cell culture experiments were linearly proportional to dose (R2 = 0.95, p < 0.0001) and correlated with IC50 across all cell lines for microdose and therapeutically relevant carboplatin concentrations (p = 0.02 and p = 0.01, respectively). A pilot microdosing clinical trial was conducted to define protocols and gather preliminary data. Plasma pharmacokinetics (PK) and [14 C]carboplatin-DNA adducts in white blood cells and tumor tissues from six NSCLC patients were quantified via AMS. The blood plasma half-life of [14 C]carboplatin administered as a microdose was consistent with the known PK of therapeutic dosing. The optimal [14 C]carboplatin formulation for the microdose was 107 dpm/kg of body weight and 1% of the therapeutic dose for the total mass of carboplatin. No microdose-associated toxicity was observed in the patients. Additional accruals are required to significantly correlate adduct levels with response.

Synthesis of two fluorescent GTPγS molecules and their biological relevance.

Fluorescent GTP analogues are utilized for an assortment of nucleic acid and protein characterization studies. Non-hydrolysable analogues such as GTPγS offer the advantage of keeping proteins in a GTP-bound conformation due to their resistance to hydrolysis into GDP. Two novel fluorescent GTPγS molecules were developed by linking fluorescein and tetramethylrhodamine to the γ-thiophosphate of GTPγS. Chemical and biological analysis of these two compounds revealed their successful synthesis and ability to bind to the nucleotide-binding site of tubulin. These two new fluorescent non-hydrolysable nucleotides offer new possibilities for biophysical and biochemical characterization of GTP-binding proteins.

DNA Adducts from Anticancer Drugs as Candidate Predictive Markers for Precision Medicine.

Biomarker-driven drug selection plays a central role in cancer drug discovery and development, and in diagnostic strategies to improve the use of traditional chemotherapeutic drugs. DNA-modifying anticancer drugs are still used as first line medication, but drawbacks such as resistance and side effects remain an issue. Monitoring the formation and level of DNA modifications induced by anticancer drugs is a potential strategy for stratifying patients and predicting drug efficacy. In this perspective, preclinical and clinical data concerning the relationship between drug-induced DNA adducts and biological response for platinum drugs and combination therapies, nitrogen mustards and half-mustards, hypoxia-activated drugs, reductase-activated drugs, and minor groove binding agents are presented and discussed. Aspects including measurement strategies, identification of adducts, and biological factors that influence the predictive relationship between DNA modification and biological response are addressed. A positive correlation between DNA adduct levels and response was observed for the majority of the studies, demonstrating the high potential of using DNA adducts from anticancer drugs as mechanism-based biomarkers of susceptibility, especially as bioanalysis approaches with higher sensitivity and throughput emerge.

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR.

Diagnostic Microdosing Approach to Study Gemcitabine Resistance.

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic "microdoses" of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of 14C-labeled gemcitabine for 4-24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity.

Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake.

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity.

This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

Cell-free expression of functional receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles.

Nanomicelle formulation modifies the pharmacokinetic profiles and cardiac toxicity of daunorubicin.

BACKGROUND: Treatment with daunorubicin (DNR) in acute myeloid leukemia is moderately effective and associated with significant side effects, including cardiac toxicity. We recently developed a nanomicellar formulation of DNR that specifically targets acute myeloid leukemia stem cells. MATERIALS & METHODS: Pharmacokinetics analysis of free DNR, DNR in nanomicellar formulations was performed in Balb/c mice and Sprague-Dawley rats. Histochemical staining, caspase 3/7, troponin and creatine kinase MB isoenzyme were used to assess toxicity. RESULTS: Compared with free DNR, the nanomicellar formulations of DNR had less cardiotoxicity as evidenced by milder histopathological changes, lower caspase 3/7 activity in heart tissue (p = 0.002), lower plasma creatine kinase MB isoenzyme (p = 0.002) and troponin concentrations (p = 0.001) postinjection. The area under curve concentration of DNR in micelles increased by 31.9-fold in mice (p < 0.0001) and 22.0-fold higher in rats (p < 0.001). CONCLUSION: Leukemia stem cell-targeting micelles dramatically change the pharmacokinetics and reduce the cardiac toxicity of DNR, which may enable improved DNR-based treatment of acute myeloid leukemia.

Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry.

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine "branches" are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6-30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.