Polyethylene Glycol Linker Research Articles

PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer.

Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.

Preclinical evaluation of a dual sstr2 and integrin αvβ3-targeted heterodimer [68Ga]-NOTA-3PEG4-TATE-RGD

PurposeMultiple receptors are co-expressed in many types of cancers. Octreotate (TATE) and Arg-Gly-Asp (RGD) peptides target somatostatin receptor 2 (sstr2) and integrin αvβ3, respectively. We developed and synthesized a heterodimer NOTA-3PEG4-TATE-RGD (3PTATE-RGD) and aimed to investigate its characteristics for dual-targeting sstr2 and integrin αvβ3. MethodsTATE and RGD peptides and 1,4,7-triazacylononane-N’,N’’,N’’’-triacetic acid (NOTA) were linked through a glutamate and polyethylene glycol (PEG) linker, then 3PTATE-RGD was labeled with 68Ga ion. Receptor-binding characteristics and tumor-targeting efficacy were tested in vitro and in vivo using H69 and A549 lung cancer cell lines and tumor-bearing mice models. Results[68Ga]-3PTATE-RGD had comparable sstr2 and integrin αvβ3-binding affinity with monomeric TATE and RGD in cell uptake and PET imaging study, respectively. In the competition study, H69 and A549 tumor uptake of [68Ga]-3PTATE-RGD was completed inhibited in the presence of an excess amount of unlabeled TATE or RGD, respectively. The blocked level didn’t grow when both of TATE and RGD mixture was co-injected with [68Ga]-3PTATE-RGD. The pharmacokinetics of [68Ga]-3PTATE-RGD is comparable with [68Ga]-TATE and [68Ga]-RGD, resulting in a larger application. Conclusion[68Ga]-3PTATE-RGD showed improved and wider tumor-targeting efficacy compared with monomeric TATE and RGD peptides, which warrants its further investigation in detection both of sstr2 and integrin αvβ3-related carcinomas.

Spatial Separation of Microbeads into Detection Levels by a Bioorthogonal Porous Hydrogel for Size-Selective Analysis and Increased Multiplexity.

Multiplex detection techniques are emerging within the fields of life science research and medical diagnostics where it is mandatory to analyze a great number of molecules. The detection techniques need to be highly efficient but often involve complicated and expensive fabrication procedures. Here, we present the immobilization and geometric separation of fluorescence-labeled microbeads for a multiplex detection in k levels. A compound of differently sized target molecules (DNA, proteins) is channeled into the respective detection levels by making use of a hydrogel as a size selective filter. The immobilized microbeads (10-20 μm) are considerably larger than the pores of the hydrogel network and therefore stay fixed at the well bottom and in higher elevations, respectively. Small biomolecules can diffuse through the pores of the network, whereas medium-sized biomolecules pass slower and large molecules will be excluded. Besides filtering, this method discriminates the used microbeads into k levels and thereby introduces a geometric multiplexity. Additionally, the exclusion of large entities enables the simultaneous detection of two target molecules, which exhibit the same affinity interaction. The hydrogel is formed through the combination of two macromonomers. One component is a homobifunctional polyethylene glycol linker, carrying a strained alkyne (PEG-BCN) and the second component is the azide-functionalized dendritic polyglycerol (dPG-N3). They react via the bioorthogonal strain-promoted azide alkyne cycloaddition (SPAAC). The hydrogel creates a solution-like environment for the diffusion of the investigated biomolecules all the while providing a stable, bioinert, and surface bound network.

Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes.

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG4-MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Anisotropic Plasmonic Metal Heterostructures as Theranostic Nanosystems for Near Infrared Light-Activated Fluorescence Amplification and Phototherapy.

The development of sophisticated theranostic systems for simultaneous near infrared (NIR) fluorescence imaging and phototherapy is of particular interest. Herein, anisotropic plasmonic metal heterostructures, Pt end‐deposited Au nanorods (PEA NRs), are developed to efficiently produce hot electrons under 808 nm laser irradiation, exhibiting the strong electric density. These hot electrons can release the heat through electron‐phonon relaxation and form reactive oxygen species through chemical transformation, as a result of potent photothermal and photodynamic performance. Simultaneously, the confined electromagnetic field of PEA NRs can transfer energy to adjacent polyethylene glycol (PEG)‐linked NIR fluorophores (CF) based on their energy overlap mechanism, leading to remarkable NIR fluorescence amplification in CF‐PEA NRs. Various PEG linkers (1, 3.4, 5.0, and 10 kD) are employed to regulate the distance between CF and PEA NRs of CF‐PEA NRs, and the maximum fluorescence intensity is achieved in CF5k‐PEA NRs. After further attachment with i‐motif DNA/Nrf2 siRNA chimera to simultaneously suppress both cellular antioxidant defense and hyperthermia resistance effects, the final biocompatible CF5k‐bPEA@siRNA NRs present promising NIR fluorescence imaging ability and 808 nm laser‐activated photothermal and photodynamic therapeutic effect in MCF7 cells and tumor‐bearing mice, holding great potential for cancer therapy.

Molecular Insight into the Protein?Polymer Interactions in N-Terminal PEGylated Bovine Serum Albumin.

Therapeutic proteins have increasingly been used in modern medical applications, but their effectiveness is limited by factors such as stability and blood circulation time. Recently, there has been significant research into covalently linking polyethylene glycol polymer chains (PEG) to proteins, known as PEGylation, to mitigate these issues. In this work, an atomistic molecular dynamics study of N-terminal conjugated PEG-BSA (bovine serum albumin) was conducted with varying PEG molecular weights (2, 5, 10, and 20 kDa) to probe PEG-BSA interactions and evaluate the effect of polymer length on dynamics. It was found that the affinity of PEG toward the protein surface increased as a function of PEG molecular weight and that a certain weight (around 10 kDa) was required to promote protein?polymer interactions. Additionally, preferential interactions were monitored through formed contacts and hotspots were identified. PEG chains coordinating in looplike conformations were found near lysine residues. Also, it was found that hydrophobic interactions played an important role in promoting PEG-BSA interactions as the PEG molecular weight increased. The results provide insight into underlying mechanisms behind transitions in PEG conformations and will aid in future design of effective PEGylated drug molecules.

A new washing-free immunosensor for tumor marker detection based on functionalized Fe3O4 submicron particles

In this submission, a novel washing-free immunosensor was designed for chemiluminescence immunoassay (CLIA) of tumor marker using human Alpha-fetoprotein (AFP) as a representative. Reactive groups were introduced on the surface of the solvothermally prepared Fe3O4 magnetic submicron particles (MSPs) and the dual-functional polyethylene glycol (PEG) linkers were modified to reduce the non-specific adsorption capacity of the particle surface and further to immobilize N-(4-aminohexyl)-N-ethylisoluminol (AHEI) and anti-AFP antibody on the surface of the particles. After incubation with AFP and horseradish peroxidase (HRP) labeled anti-AFP antibody respectively, the formed sandwich-type immunocomplexes on the particle surface could lead to the close proximity between AHEI and HRP, thus eventually leads to an increase in the detected chemiluminescence (CL) signal. As a bound HRP labeled anti-AFP antibody produces much stronger CL signal than an unbound one, this approach eliminates the time- and labor-consuming washing steps usually required of such assays. Under the optimal conditions, this present method shows a wide quantitative range from 1 to 250 ng mL−1 with a detection limit of 1.751 ng mL−1. The detection limit was sufficient for measuring physiological (0–20 ng mL−1) and clinical (>20 ng mL−1) AFP levels. This method can also be applied in serum samples, and also avoid the problem of particle aggregation caused by multiple separation and washing steps.

Characterization of Polyethylene Glycol-Reinforced Alginate Microcapsules for Mechanically Stable Cell Immunoisolation.

Islet transplantation within mechanically stable microcapsules offers the promise of long-term diabetes reversal without chronic immunosuppression. Reinforcing the ionically gelled network of alginate (ALG) hydrogels with covalently linked polyethylene glycol (PEG) may create hybrid structures with desirable mechanical properties. This report describes the fabrication of hybrid PEG-ALG interpenetrating polymer networks and the investigation of microcapsule swelling, surface modulus, rheology, compression, and permeability. It is demonstrated that hybrid networks are more resistant to bulk swelling and compressive deformation and display improved shape recovery and long-term resilience. Interestingly, it is shown that PEG-ALG networks behave like ALG during microscale surface deformation and small amplitude shear while exhibiting similar permeability properties. The results from this report's in vitro characterization are interpreted according to viscoelastic polymer theory and provide new insight into hybrid hydrogel mechanical behavior. This new understanding of PEG-ALG mechanical performance is then linked to previous work that demonstrated the success of hybrid polymer immunoisolation devices in vivo.

Conjugation of prostate cancer-specific aptamers to polyethylene glycol-grafted polyethylenimine for enhanced gene delivery to prostate cancer cells

Prostate cancer is one of the most frequently diagnosed cancers. In this study, a prostate cancer-specific aptamer, Wy5a, was conjugated to polyethylenimine (PEI) with a polyethylene glycol (PEG) linker for prostate cancer-specific gene delivery. PEI-PEG-AS1411 was synthesized as a non-specific control. PEI-PEG-Wy5a formed complexes with plasmid DNA in a spherical form with a diameter of 70nm and a positive surface-charge. In transfection assays, PEI-PEG-Wy5a had higher gene delivery efficiency to PC-3 prostate cancer cells than did PEI-PEG or PEI-PEG-AS1411, but not in other types of cells. The results suggest that PEI-PEG-Wy5a may be useful for prostate cancer-specific gene delivery.

Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators.

Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.

Site-specific labeling of an anti-MUC1 antibody: probing the effects of conjugation and linker chemistry on the internalization process.

Antibody-drug conjugates (ADCs) have recently received enormous attention as an attractive approach for cancer therapy. Although ADC design has been believed to be important for the relative efficacy of ADCs, it remains unexplored how the structural characteristics of ADCs would impact the internalization process and intracellular trafficking of the molecules. Herein, we report our efforts in investigating the cellular endocytosis implications of the conjugation and linker chemistry in designing antibody-based agents. A series of anti-MUC1 single-chain variable fragment (scFv-SM3) conjugates were designed with unique structural characteristics ranging from conjugation methods, sites of attachment and linker chemistry. In vitro confocal imaging showed that both random lysine-conjugation and site-specific conjugation, including C-terminus modification or internal site conjugation, could afford antibody conjugates with similar binding affinity and cellular uptake to target-expressing cells. Time-course internalization studies demonstrated that SM3-conjugates with short polyethylene glycol linkers outcompeted those that lack any hydrophilic linkers for higher cellular uptake and faster internalization rate. The SM3-conjugates with the highest affinity and internalization rate were also tested in mouse xenograft models using MUC1-overexpressing tumor cells. Our results indicate that the linker and conjugation chemistry play an important role in the internalization process of antibody conjugates, and this in turn could impact the therapeutic effects of ADCs.

Triple Degradation of BTK, IKZF1 and IKZF3 in B-Cell Malignancies

Bruton's Tyrosine Kinase (BTK), a TEC-family non-receptor tyrosine kinase, plays a critical role in B-cell development and function. Targeting of BTK with covalent inhibitors like ibrutinib has become a standard approach for many B-cell malignancies, including chronic lymphocytic leukemia (CLL), Waldenstrom macroglobulinemia, mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). Yet, many patients demonstrate intrinsic or acquired resistance to covalent BTK inhibition. The prognosis of patients who relapse after ibrutinib treatment for MCL or CLL is dismal, highlighting the urgent need for new approaches that overcome resistance to current BTK inhibitors. We hypothesized that degradation of BTK could be a better alternative to inhibition alone, as it would both: 1) maintain efficacy in cells harboring the ibrutinib-resistant BTK C418S mutation and 2) target non-catalytic functions of BTK. To address this, we turned to a small molecule-mediated protein degradation platform that utilizes an E3 ligase-targeting moiety linked to the ligand of a target of interest, so that the target can be marked for ubiquitination and subsequent proteasomal degradation. We previously showed that BTK is one of the most robustly degraded kinase targets using a nonspecific kinase inhibitor linked to a imide-based core followed by agnostic proteomics. We synthesized highly potent and selective degraders of BTK using imides as a base. To do so, the parent BTK inhibitor CGI1746 was linked to thalidomide using either polyethylene glycol (DD-03-007) linkers or saturated hydrocarbon chain (DD-03-171) linkers. After verifying that these degraders induce dimerization of BTK and CRBN and penetrate cells, we explored the pharmacological effects in vitro. Both DD-03-007 and DD-03-171 reduced BTK levels at concentrations as low as 40 nM within 4h of treatment. Furthermore, cellular BTK levels remained low for 24h after washout, showing that these degraders are capable of sustaining depletion of BTK for an extended period of time (Figure). DD-03-007 and DD-03-171 are both ligase and proteasome-dependent, as shown by co-treatment with bortezomib or MLN-4924 as well as competition experiments with lenalidomide or CGI-1746. Moreover, the degraders exhibited strong synergy with the HCK inhibitor A419259, suggesting that it would be possible to recapitulate ibrutinib's previously reported polypharmacology with an HCK inhibitor.We overexpressed wild-type or C481S BTK in TMD-8 cells and ran an antiproliferation assay, which showed that DD-03-007 overcame ibrutinib resistance associated with BTK C481S mutation.Next, we explored the effects of the degraders in MCL specifically. Proteomic analysis showed that DD-03-171 is a triple-degrader, as it degrades BTK but retains degradation activity on IKZF1 and IKZF3. Finally, we performed In vivo efficacy studies in cell line and patient-derived xenograft (PDX) models of MCL. The latter was obtained from a patient who had progressed on ibrutinib. In both models, DD-03-171 caused a significant reduction in tumor burden at an early timepoint. DD-03-171 also markedly extended survival compared to treatment with ibrutinib or lenalidomide alone (Figure). In conclusion, we developed highly potent and selective BTK degraders with activity in vivo against human MCL that induce the degradation of multiple factors essential for MCL survival. [Display omitted] DisclosuresTreon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Johnson & Johnson: Consultancy; BMS: Research Funding. Weinstock:Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership.

Integrin αvβ3-Targeted [64Cu]CuS Nanoparticles for PET/CT Imaging and Photothermal Ablation Therapy.

Copper sulfide (CuS) nanoparticles have been considered one of the most clinical relevant nanosystems because of their straightforward chemistry, small particle size, low toxicity, and intrinsic theranostic characteristics. In our previous studies, radioactive [64Cu]CuS nanoparticles were successfully developed to be used as efficient radiotracers for positron emission tomography and for photothermal ablation therapy of cancer cells using near-infrared laser irradiation. However, the major challenge of CuS nanoparticles as a theranostic platform is the lack of a means for effective targeted delivery to the tumor site. To overcome this challenge, we designed and synthesized angiogenesis-targeting [64Cu]CuS nanoparticles, which are coupled with cyclic RGDfK peptide [c(RGDfK)] through polyethylene glycol (PEG) linkers using click chemistry. In assessing their tumor-targeting efficacy, we found that the tumor uptakes of [64Cu]CuS-PEG-c(RGDfK) nanoparticles at 24 h after intravenous injection were significantly greater (8.6% ± 1.4% injected dose/gram of tissue) than those of nontargeted [64Cu]CuS-PEG nanoparticles (4.3% ± 1.2% injected dose/gram of tissue, p < 0.05). Irradiation of tumors in mice administered [64Cu]CuS-PEG-c(RGDfK) nanoparticles induced 98.7% necrotic areas. In contrast, irradiation of tumors in mice administered nontargeted CuS-PEG nanoparticles induced 59% necrotic areas ( p < 0.05). The angiogenesis-targeting [64Cu]CuS nanoparticles may serve as a promising platform for image-guided ablation therapy with high efficacy and minimal side effects in future clinical translation of this novel class of multifunctional nanomaterials.

Targeting reactive oxygen species for respiratory infection: Fact or fancy?

Targeting reactive oxygen species for respiratory infection: Fact or fancy?

Liposomal codelivery of an SN38 prodrug and a survivin siRNA for tumor therapy.

PurposeA liposome-based siRNA–drug combination was evaluated as a potential therapeutic strategy to improve the curative effect.MethodsA topoisomerase inhibitor SN38 prodrug was combined with a survivin siRNA through codelivery using transferrin (Tf)-L-SN38/P/siRNA. In this combination, SN38 was conjugated to the cell penetrating peptide TAT through a polyethylene glycol (PEG) linker to synthesize TAT-PEG-SN38. The amphiphilic TAT-PEG-SN38 was used as an ingredient of liposomes to improve the cellular uptake. Protamine was added to form an electrostatic complex with siRNA in the core of the liposomes. Tf was introduced to enable tumor cell targeting of liposomes (Tf-L-SN38/P/siRNA).ResultsTf-L-SN38/P/siRNA exhibited a particle size of 148 nm and a ζ-potential of +7.8 mV. The cellular uptake and antitumor activity were dependent on Tf receptor targeting, TAT-PEG-SN38, and siRNA codelivery. Tf-L-SN38/P/siRNA was shown to be considerably more effective than liposomes carrying individual components. This combination induced potent tumor inhibition (76.8%) in HeLa cell xenograft tumor-bearing nude mice.ConclusionThese data indicated that Tf-L-SN38/P/siRNA was an effective system for codelivery of SN38 and a survivin siRNA and that its therapeutic potential deserved further evaluation.

A biotin receptor-targeted silicon(IV) phthalocyanine for in vivo tumor imaging and photodynamic therapy

Phthalocyanines (Pcs) are a kind of potential photosensitizers for fluorescence imaging and photodynamic therapy (PDT). However, the clinical application of Pcs is suffered from their poor solubility, high aggregation tendency and low tumor-specificity. To address these problems, two biotin moieties were linked to the axial positions of silicon(IV) phthalocyanine (SiPc) through hydrophilic polyethylene glycol (PEG) linkers to synthesize a new water-soluble and tumor-targeting photosensitizer (compound 1). The introduction of PEG linkers on SiPc markedly reduced the aggregation tendency of the conjugate. In vitro assays also proved that compound 1 could specifically accumulate in biotin receptor (BR) positive Hela cells through the BR-mediated internalization. Owing to the good characteristics of water-solubility and low aggregation, the bioactivity of compound 1 was examined in the xenograft tumor model. In vivo imaging and tissue distribution studies showed that compound 1 selectively accumulated in the tumor tissue, with tolerable signals found in other organs of the tumor-bearing mice. Furthermore, compound 1 could significantly depress tumor progression in vivo under irradiation. After 14 days of the treatment, the tumor volumes were even smaller than the beginning size. All these results reveal that compound 1 is a promising candidate, with low aggregation tendency, high tumor-specificity and water-solubility, for in vivo tumor diagnosis and PDT treatment.

Cisplatin-loaded hollow gold nanoparticles for laser-triggered release

BackgroundHollow gold nanoparticles (HGNPs) exposed to near-infrared (NIR) light yield photothermal effects that can trigger a variety of biological effects for potential biomedical applications. However, the mechanism of laser-triggered drug release has not been studied before.MethodsA tripeptide Ac-Glu-Glu-Cys-NH2 (Ac-EEC) was directly linked to the surface of HGNPs. The EEC-HGNPs conjugate was then complexed with cisplatin Pt(II) to give Ac-EEC(Pt)-HGNPs. Folic acid was introduced to the gold surface of Ac-EEC-HGNPs through a thioctic acid-terminated polyethylene glycol linker (F-PEG-TA) followed by complexation with Pt(II) to give F-Ac-EEC(Pt)-HGNPs. Laser treatment was instituted with a 15-ns pulsed laser at a repetition rate of 10 Hz. The released Pt(II) was quantified by inductively coupled plasma mass spectroscopy, and the nature of the released Pt-containing species was characterized by liquid chromatography–mass spectroscopy. The cytotoxicity was studied using the MTT assay.ResultsPt(II) was released from Ac-EEC(Pt)-HGNPs via two modes: (1) sustained release through an inverse ligand exchange reaction with chloride ions and (2) rapid release through cleavage of the Au–S bond between the tripeptide linker and Au surface upon NIR laser irradiation. The folate (F) conjugate of the nanoconstruct, F-Ac-EEC(Pt)-HGNPs, in combination with laser treatment showed a significantly greater effect on cell mortality against folate-overexpressing human epidermoid carcinoma KB cells than F-Ac-ECC(Pt)-HGNPs alone after 24 h of incubation.ConclusionsThese results demonstrate that the photothermal property of HGNPs can be used for dual-modality photothermal therapy and NIR laser-triggered platinum-based chemotherapy.

Uptake Pathways of Protein-Coated Magnetic Nanoparticles in Platelets.

Magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. Platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (HSA). However, the optimal HSA density coated on particles and the uptake mechanism of single particles in platelets remain unclear. Here, we utilized single-molecule force spectroscopy (SMFS) and other complementary methods to characterize the interaction of particles when interacting with platelets and to determine the optimal HSA amount required to coat particles. An HSA concentration of 0.5-1.0 mg/mL for coating particles is most efficient for platelet labeling. Binding pathways could be elucidated by linking a single HSA particle to SMFS tips via polyethylene glycol (PEG) linkers of different lengths and allowing them to interact with immobilized platelets on the substrate. Depending on the PEG length (i.e., short ∼2 nm, medium ∼30 nm, and long ∼100 nm), particles interact differently with platelets as shown by one, two, or three force distributions, which correspond up to three different binding pathways, respectively. We propose a model that the short PEG linker allows the particle to interact only with the platelet membrane, whereas the medium and long PEG linkers promote the particle to transfer from open canalicular system to another target inside platelets. Our study optimizes magnetic platelet labeling and provides details of particle pathways in platelets.

Abstract 755: Synthesis and evaluation of sugar alcohol based molecules for drug conjugation and delivery

Abstract Hydrophilic linkers, which are available in different motifs, have been used as agents of drug delivery, as aqueous solubility enhancers for hydrophobic molecules, and as tethers or spacers in conjugation. Polyethylene glycol (PEG) is one of the most commonly used hydrophilic linkers and commercially available. Lower molecular weight (MW) heterobifunctional PEG linkers are the most frequently used linkers for linking hydrophobic compounds, whereas high MW polymers have been used to conjugate proteins, peptides, oligonucleotides, siRNA, and other therapeutic biopolymers. Here, we describe a new type of biomaterial based on sugar alcohols: AqueaTether™ (AqT™) molecules. Various AqT™ linkers with different lengths and end groups were synthesized and their hydrophilic nature and ability to enhance the water solubility of hydrophobic compounds compared to different commercial and PEG linkers. Various antibody drug conjugates (ADCs) with water soluble AqT™-drugs were prepared. An ADC made with AqT™-monomethyl auristatin E (MMAE) with only two sugar alcohol units attached and an average drug-to-antibody ratio (DAR) of 4 demonstrated significant improvements in minimizing the aggregation of antibodies and long-term stability during storage. Due to increased water solubility, AqT™ linkers also allow higher loading of drugs per antibody, enabling the use of less toxic drugs, potentially increasing the overall efficacy and safety profile of an ADC. For example, ADCs with average DARs of 8 and 15 have been made using AqT™-doxorubicin with only two sugar alcohol units attached. In peptide-based drug discovery, discrete high MW AqT™ linker is used to label peptide, resulting in an increased half-life in human serum. In summary, AqT™ molecules are by far the most hydrophilic and versatile linkers currently in development. AqT™ molecules represent a great tool for drug conjugation and delivery. Citation Format: Yumei Huang, Shuyan Chen, Binyuan Sun, Amanda Griffin, Nathanial Sweet, Mingqian Feng, Mitchell Ho. Synthesis and evaluation of sugar alcohol based molecules for drug conjugation and delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 755.

Abstract 3707: PD-L1 antibody drug conjugate for cancer immune-chemo combination therapy

Abstract Antibody drug conjugates (ADCs) are a new form of targeted therapy to cancer, consisting of an antibody, a conditionally stable linker, and a cytotoxic drug. Immune checkpoint molecule, such as programmed death ligand-1 (PD-L1) overexpressed in various solid tumors including triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) and it has a crucial role in tumor immune evasion. Blocking the interaction between PD-L1 and PD-1 (programmed death-1 receptor) resurrects tumor immune surveillance, resulting CD8+ cytotoxic T cells activation to attack the tumor cells. This is an emerging anticancer mechanism and more than four antibody inhibitors have been recently FDA approved against PD-1 or PD-L1 biomarker. Targeting PD-L1 in TNBC and NSCLC is an excellent approach for tumor immunotherapy. However, the major challenges of anti-PD-L1 therapy (Atezolizumab) are (i) the transient expression of PD-L1 in tumor microenvironment, (ii) development of anti-PD-L1 resistance, (iii) difficulty of tumor stroma penetration that retard its clinical outcome. To overcome these challenges, we chemically conjugated Atezolizumab (ATZ) with potent chemotherapeutic payload, such as Doxorubicin (Dox) through polyethylene glycol (PEG) linkers. The role of Dox is to break the tumor stroma so that PD-L1 antibody can penetrate to the core of the tumor. Introduction of PEG-linker with high molecular weight ATZ (155 kD) has drastically improved the solubility of ADC and successful chemical conjugation of between ATZ and PEG-Dox was evaluated by MALDI-TOF analysis. Our data has revealed significantly tumor cell killing effect and induction of apoptosis in PD-L1-Dox treated MDA-MB-231 cells. The near infrared imaging of PD-L1 dye conjugate in TNBC and NSCLC patient derived xenograft (PDx) supports the selective tumor targetability. Based on the synergistic anticancer effect and selective tumor targetability demonstrated in this study build a strong rational of using PD-L1 ADC approach for cancer immune-chemo therapy and diagnosis. Citation Format: Samaresh Sau, Alex Petrovici, Hashem Alsaab, Arun K. Iyer. PD-L1 antibody drug conjugate for cancer immune-chemo combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3707.