Adem Yildirim

Self-assembled peptide amphiphile (PA)-based nanostructures are promising biomaterials with their biocompatibility and highly tunable physical and chemical properties. Despite this promise, the interactions of PA nanostructures with biological systems have remained largely unexplored. Here, we investigate nano–bio interactions of self-assembled PA nanostructures using a series of PAs with varying amphiphilicities, achieved by tuning the hydrophilicity of the peptide backbone and payloads attached to them. We show that, while PAs with hydrophilic peptide backbones and payloads mostly disassembled in blood plasma, increasing overall hydrophobicity improved their stability. We also report that disassembled PAs can bind to blood biomolecules and binding shifted from albumin to lipoproteins with increasing hydrophobicity. In addition, we show that the membrane-binding affinity of PAs increases with increasing PA hydrophobicity, which also induces membrane disruption and cytotoxicity. The results of this study highlight the critical role of the overall amphiphilic balance of PAs on their interactions with biological molecules and cell membranes.

Understanding the interactions of nanomaterials with biological systems is essential to designing effective nanomedicines. However, most of our understanding originates from studies with solid nanoparticles, and nano‐bio interactions of self‐assembled nanomaterials have remained largely unexplored. To address this knowledge gap, we develop a series of self‐assembling peptide amphiphiles (PAs) with different lipid modifications and investigate their interactions with biological systems. We find that PA nanostructures rapidly disassemble and reassemble with albumin and lipoproteins in blood plasma. While PAs with shorter lipid tails mainly assemble with albumin, increasing lipid length shifts binding to lipoproteins. All PAs show strong tumor accumulation in 4T1 tumor‐bearing mice with tumor to liver ratios of ∼3–6. Overall, albumin‐binding improves blood circulation (up to 1.6‐fold) and tumor accumulation (up to 3‐fold) compared to lipoprotein‐binding, but also shows slower healthy tissue clearance. This study shows that the biointeractions of self‐assembled nanomaterials can be controlled through molecular design, which may lead to the development of effective nanomedicines.

Lipid-based formulations (liposomes, micelles, lipid nanoparticles, emulsions, lipid prodrugs) are the most popular systems for tumor drug delivery. At the same time, there is limited knowledge of the factors controlling the lipid structure-tumor accumulation relationship (STAR). To address this question, we synthesized a compact library of lipids with the shared cyanine Cy3 headgroup but variable tail hydrophobicity and headgroup-tail linkers. A shared fluorophore enabled the straightforward comparison of pharmacokinetics, tumor accumulation, and interactions of lipids with serum and cells. The library was formulated into nanomicelles with DSPE-PEG2000 and screened for tumor accumulation after intravenous injection in the syngeneic 4T1 breast cancer mouse model. Cy3 lipids with ester linkers mostly displayed poor tissue and tumor accumulation, except Cy3-cholesterol. Cy3 lipids with amide linkers and indocarbocyanine derivatives of Cy3 (DiI) showed better tumor accumulation. Nonlipid molecules Cy3-COOH and Cy3-PEG5000 were rapidly cleared with minimal accumulation in tumors. Of all lipids, DiI-C18 and DiI-C22 showed superior accumulation in 4T1 breast, GL261, and CT-2A orthotopic glioma models. Subsequent investigation revealed that chemical and formulation instability negatively affect the lipid pharmacokinetics and tumor accumulation. Lipids with stable linkers and hydrophobic chains caused slow clearance and high tumor buildup. On the other hand, short-chain lipids showed increased interaction with low-density lipoprotein (LDL), with strong evidence indicating accelerated clearance by the liver LDL receptor. The uptake of short-chain lipids by tumor cells in vitro was inhibited by interaction with lipoproteins. Overall, serum stability and lipoprotein interaction emerged as important in vitro predictors of favorable pharmacokinetics and tumor accumulation. These findings provide a framework for designing effective lipid-based therapeutics and imaging agents.

The interactions of nanomaterials with biomolecules in vivo determine their biological fate. Here, it is shown that self‐assembled peptide amphiphile (PA) nanostructures can dynamically interact with endogenous biomolecules and take advantage of naturally occurring processes to target a broad range of solid tumors. In circulation, self‐assembled PA nanostructures disassemble and reassemble mainly with lipoproteins, which prolongs blood circulation and dramatically improves tumor accumulation and retention. Mechanistic studies suggested that PAs internalize into cancer cells by assembling with their cell membranes and independently of specific receptors. By exploiting these interactions, a PA developed in this study (namely Self‐Assembly ‐ Glutamic acid, SA‐E) demonstrates specific accumulation in various xenograft, syngeneic, patient‐derived xenograft, or transgenic rodent models. In addition, SA‐E enabled the effective delivery of highly potent chemotherapy to different syngeneic and xenografted tumors with reduced side effects. With its simple and modular design and universal tumor accumulation mechanism, SA‐E represents a promising platform for broad applications in cancer imaging and therapy.

Mechanical tumor ablation using focused ultrasound (FUS) offers minimally invasive ablation of solid tumors. However, mechanical tumor ablation requires highly intense FUS pulses, which may generate off-target effects. In addition, residual cancer cells that survive ablation can cause recurrence. To overcome these challenges, we developed a dual-functional nanomaterial by assembling peptide amphiphiles on hydrophobically modified nanoparticles. The hydrophobic nanoparticle core allows for the generation of cavitating bubbles at low FUS intensities to effectively ablate tumors. Peptide amphiphile shells interact dynamically with the hydrophobic nanoparticle surfaces and cell membranes, which improved tumor retention and cellular internalization of payloads attached to them. By conjugating a highly potent agent, Monomethyl auristatin E, to the peptide amphiphiles, we showed synergistic mechano- and chemotherapy using in vitro and in vivo models of human melanoma. This work presents a new nanoparticle approach to improving the outcomes of mechanical tumor ablation.

Proteases are promising biomarkers for cancer early detection. Their enzymatic activity against peptide substrates allows for their straightforward detection using low-cost tests. However, the complexity of the human proteome makes it challenging to develop sensitive and selective tests against a specific protease biomarker. Here, we report a different approach by utilizing the total protease activity in plasma samples to detect pancreatic cancer. Instead of targeting a specific protease using a specific peptide substrate, we utilized an array of 360 FRET substrates to screen for cleavage patterns in plasma samples collected from screen negatives and pancreatitis or pancreatic ductal adenocarcinoma cancer (PDAC) patients. In this proof of concept study, we first screened all 360 substrates using a small cohort (n = 13) to identify the top 5 substrates that best separate different conditions. Then, we performed a validation study using a larger cohort (n = 86) and the selected substrates. There was a statistically significant increase in the total protease activity in PDAC samples compared to screen negative and pancreatitis samples. The selected substrates detected PDAC with an area under the curve (AUC) of 0.8. This work represents a novel strategy for identifying peptide substrates for the detection of PDAC and other cancers.

The interactions of nanomaterials with biomolecules in vivo determine their biological fate. Here, we show that self-assembled peptide amphiphile (PA) nanostructures can dynamically interact with endogenous biomolecules and take advantage of naturally occurring processes to target a broad range of solid tumors. In circulation, self-assembled PA nanostructures disassemble and reassemble mainly with lipoproteins, which prolongs blood circulation and dramatically improves tumor accumulation and retention. Mechanistic studies suggested that PAs internalize into cancer cells by assembling with their cell membranes and independently of specific receptors. By exploiting these interactions, a PA developed in this study (namely SA-E) demonstrated specific accumulation in various xenograft, syngeneic, patient-derived xenograft, or transgenic rodent models. In addition, SA-E enabled the effective delivery of highly potent chemotherapy to different syngeneic and xenografted tumors with reduced side effects. With its simple and modular design and universal tumor accumulation mechanism, SA-E represents a promising platform for broad applications in cancer imaging and therapy.

Localization-based ultrasound imaging methods that use microbubbles or nanodroplets offer high-resolution imaging with improved sensitivity and reduced background signal. However, these methods require long acquisition times (typically seconds to minutes), preventing their use for real-time imaging and, thus, limiting their clinical translational potential. Here, we present a new ultrafast localization method using blinking ultrasound-responsive nanoparticles (BNPs). When activated with high frame rate (1 kHz) plane wave ultrasound pulses with a mechanical index of 1.5, the BNPs incept growth of micrometer-sized bubbles, which in turn collapse and generate a blinking ultrasound signal. We showed that background-free ultrasound images could be obtained by localizing these blinking events using acquisition times as low as 11 ms. In addition, we demonstrated that BNPs enable in vivo background-free ultrasound imaging in mice. We envision that BNPs will facilitate the clinical translation of localization-based ultrasound imaging for more sensitive detection of cancer and other diseases.

Surface‐engineered hydrophobic nanoparticles that can stabilize small gas pockets on their surfaces (i.e., gas‐stabilizing nanoparticles, GSNs) have been recently shown to be excellent contrast and cavitation agents for ultrasound theranostics. However, previously developed GSNs are not biodegradable, which limits their clinical translation potential. Here the development of biodegradable GSNs is shown by coating hydrophobically modified mesoporous silica nanoparticles with different protein solutions. It is found that these novel GSNs retain strong cavitation activity while rapidly degrading in simulated body fluid (SBF) or in vivo in days or a few weeks, respectively. Interestingly, GSNs coated with other stabilizing layers, Pluronic F127 polymer or phospholipids, demonstrated significantly slower degradation rates with only partial degradation even after a month of incubation in SBF. Next, it is shown that these biodegradable GSNs can be used to ablate tumor xenografts at lower ultrasound intensities, thus avoiding the side effects of high‐intensity ultrasound. Finally, it is shown that only tumors treated with GSNs and ultrasound can specifically enrich for circulating tumor DNA, which will improve liquid biopsies for understanding tumor heterogeneity and treatment response. Overall, this study details a simple yet effective method for preparing biodegradable GSNs with broad potential for applications in cancer diagnosis and therapy.

The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting spike (S) protein cleavage by several proteases. We developed a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.