Glycol Linker Research Articles

Immunogenicity assessment strategy for a chemically modified therapeutic protein in clinical development.

The clinical immunogenicity assessment for complex multidomain biological drugs is challenging due to multiple factors that must be taken into consideration. Here, we describe a strategy to overcome multiple bioanalytical challenges in order to assess anti-drug antibodies (ADA) for a novel and unique chemically modified protein therapeutic. A risk-centered approach was adopted to evaluate the immunogenic response to a modified version of human growth differentiation factor 15 (GDF15) connected to an albumin-binding fatty acid via a polyethylene glycol (PEG) linker. Key steps include monitoring anti-drug antibodies (ADAs), using a standard tiered approach of screening and confirmation. To deepen our understanding of ADA response, as a third tier of immunogenicity assessment, novel extensive characterization using a set of assays was developed, validated, and used routinely in clinical sample analysis. This characterization step included performance of titration, mapping of ADA response including anti-GDF15 and anti-PEG-fatty-acid antibody characterization, and assessment of the neutralizing anti-drug antibodies (NAbs) using cell-based assays for immunogenicity in parallel. The analytical methods were applied during two clinical trials involving both healthy volunteers and overweight or obese patients. We observed low incident rates for ADA and no ADAs against the PEG linker with fatty acid conjugation. In one of the clinical studies, we identified neutralizing ADAs. The proposed novel strategy of extensive characterization proved effective for monitoring the presence of ADAs and NAbs and can be used to support clinical development of a broad range of chemically modified proteins and multidomain biotherapeutics.

In Situ Self-Assembly of Antibody-Rhamnose Complex as a Pre-Targeting Strategy for Enhanced Cancer Immunotherapy.

Enhancing the Fc effector functions of monoclonal antibodies (mAbs) is a proven strategy for improving cancer immunotherapy. In this study, we present a novel pre-targeting approach that integrates host-guest chemistry with an antibody-recruiting concept to create mAbs with superior effector functions. Using rituximab (RTX), a clinically approved anti-CD20 mAb, as our model, we modified RTX by conjugating it with adamantane (Ada) derivatives and various polyethylene glycol (PEG) linkers to produce RTX-Ada conjugates. These conjugates effectively formed RTX-rhamnose (Rha) complexes in situ through self-assembly, driven by host-guest interactions with Rha-modified β-cyclodextrin. This mechanism successfully redirected endogenous anti-Rha antibodies to target cells, enhancing the availability of Fc domains for improved effector functions, including complement-dependent cytotoxicity (CDC). A structure-activity relationship study indicated that the potency of these in situ complexes was significantly influenced by the length of the PEG linker used; shorter PEG linkers correlated with higher CDC activity. Given the variability in endogenous antibody levels among individuals, this strategy presents a flexible and promising platform for enhancing the efficacy of mAb-based cancer immunotherapy.

Dual-Modality Imaging with a Zwitterionic Fluorescent Probe for Reversible Monitoring of Mitochondrial Membrane Potential Dynamics.

Traditional fluorescence intensity-based probes face challenges in accurately measuring mitochondrial membrane potential (MMP) due to intramolecular fluorescence quenching. In this work, we introduce a novel approach by incorporating quenching moieties within the zwitterionic probe to eliminate self-quenching interference, thus, enabling real-time and precise visualization of reversible MMP changes. We synthesized a zwitterionic fluorescent probe consisting of silicon-rhodamine (SiR) that was hydroxyl-substituted on the bay position of perylene diimides (PDIs) connected via a polyethylene glycol (PEG) linker. The lipophilic cationic SiR facilitates the entry of the PDI into the mitochondria, where the alkaline pH environment (pH = 8.0) ionizes the hydroxyl to a negatively charged species, affecting the quenching efficiency of SiR depending on the distance between the PDI and SiR moieties regulated by the MMP. The rigid aromatic ring of the PDI and strong hydrophobic interactions with the lipid bilayer, along with the inhibitory effect of the negatively charged hydroxyl on internalization, ensure the retention of PDI within the mitochondria. As the MMP decreases, SiR shifts outward, reducing quenching by phenolic anions and restoring fluorescence. Conversely, as the MMP increases, SiR moves inward, intensifying quenching by phenolic ions and reducing fluorescence, enabling reversible visualization monitoring of the MMP. This strategy overcomes the limitations of traditional intensity-based probes, providing a new avenue for reversible monitoring of the MMP.

Coumarin-Based Photodegradable Hydrogels Enable Two-Photon Subtractive Biofabrication at 300 mm s-1.

Spatiotemporally controlled two-photon photodegradation of hydrogels has gained increasing attention for high-precision subtractive tissue engineering. However, conventional photolabile hydrogels often have poor efficiency upon two-photon excitation in the near-infrared (NIR) region and thus require high laser dosage that may compromise cell activity. As a result, high-speed two-photon hydrogel erosion in the presence of cells remains challenging. Here we introduce the design and synthesis of efficient coumarin-based photodegradable hydrogels to overcome these limitations. A set of photolabile coumarin-functionalized polyethylene glycol linkers are synthesized through a Passerini multicomponent reaction. After mixing these linkers with thiolated hyaluronic acid, semi-synthetic photodegradable hydrogels are formed in situ via Michael addition crosslinking. The efficiency of photodegradation in these hydrogels is significantly higher than that in nitrobenzyl counterparts upon two-photon irradiation at 780 nm. A complex microfluidic network mimicking the bone microarchitecture is successfully fabricated in preformed coumarin hydrogels at high speeds of up to 300 mm s-1 and low laser dosage down to 10 mW. Further, we demonstrate fast two-photon printing of hollow microchannels inside a hydrogel to spatiotemporally direct cell migration in 3D. Collectively, these hydrogels may open new avenues for fast laser-guided tissue fabrication at high spatial resolution.

Salt-Induced Linker Dehydration Modulates Micellar Structure in Ether-Linked Sulfate Surfactants.

Ether-linked surfactants are widely used in formulations such as liquid soaps, but despite their ubiquity, it is unclear how n-ethylene glycol linkers in surfactants, such as sodium lauryl n-(ethylene glycol) sulfate (SLEnS), influence micellar packing in the presence of NaCl. In the present work, we probe the structure and hydration of ether linkers in micelles comprising monodisperse SLEnS surfactants using contrast-variation small-angle neutron scattering (CV-SANS) and small-angle X-ray scattering (SAXS). Using SAXS, changes in micellar structure were observed for SLEnS (n = 1, 2, or 3) arising from the extent of ethoxylation. Scattering profiles indicated a clear transition from elongated cylindrical micelles to shorter ellipsoidal micelles with increasing ethoxylation. With CV-SANS, micellar structure and linker geometries of SLE3S were able to be resolved, indicating that a change in micellar architecture is modulated by dehydration of the tri(ethylene glycol) linker, offering new insights into the role of water and ions in the self-assembly of this key class of surfactants.

Development of a Stable Peptide-Major Histocompatibility Complex (MHC) via Sortase and Click Chemistry.

T cells play a crucial role in antitumor immune responses and the clearance of infected cells. They identify their targets through the binding of T-cell receptors (TCRs) to peptide-major histocompatibility complex (pMHC) molecules present in cancer cells, infected cells, and antigen-presenting cells. This interaction is often weak, requiring multimeric pMHC molecules to enhance the avidity for identifying antigen-specific T cells. Current exchangeable pMHC-I tetramerization methods may overlook TCRs recognizing less stable yet immunogenic peptides. In vivo applications targeting antigen-specific T cells demand the genetic synthesis of a pMHC fusion for each unique peptide antigen, which poses a significant challenge. To address these challenges, we developed a sortase and click chemistry-mediated approach for generating stable pMHC molecules. Leveraging sortase technology, we introduced an azide click-handle near the N-terminus of β2m, proximal to the MHC-peptide-binding groove. Simultaneously, the peptide was engineered with a multi glycine linker and a C-terminal alkyne click-handle. Azide-alkyne click reactions efficiently immobilized the peptide onto the MHC molecule, providing a versatile and efficient method for pMHC generation. The resulting peptide-clicked-MHC specifically binds to its cognate TCR and remains stable for over 3 months at 4 °C in the absence of any additional free peptide. The stability of the pMHC and its affinity to cognate TCRs are influenced by the linker's nature and length. Multi glycine linkers outperform poly(ethylene glycol) (PEG) linkers in this regard. This technology expands the toolkit for identifying and targeting antigen-specific T cells, enhancing our understanding of cancer-specific immune responses, and has the potential to streamline the development of personalized immunotherapies.

Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation.

Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fcγ receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fcγ interactions with the goal of driving an effective antitumor immune response, including Fc point mutations and glycan modifications. However, robust antibody-Fcγ engagement and immune cell binding of Fc-enhanced antibodies in the periphery can lead to the unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Creating a balance between effective engagement of Fcγ receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Herein, we describe a method for the reversible chemical modulation of antibody-Fcγ interactions using simple poly(ethylene glycol) (PEG) linkers conjugated to antibody interchain disulfides with maleimide attachments. This method enables dosing of a therapeutic with muted Fcγ engagement that is restored in vivo in a time-dependent manner. The technology was applied to an effector function enhanced agonist CD40 antibody, SEA-CD40, and experiments demonstrate significant reductions in Fc-induced immune activation in vitro and in mice and nonhuman primates despite showing retained efficacy and improved pharmacokinetics compared to the parent antibody. We foresee that this simple, modular system can be rapidly applied to antibodies that suffer from systemic immune activation due to peripheral FcγR binding immediately upon infusion.

Liposomal STAT3-Degrading PROTAC Prodrugs Promote Anti-Hepatocellular Carcinoma Immunity via Chemically Reprogramming Cancer Stem Cells.

Cancer stem cells (CSCs) with hyperactivated signal transducer and activator of transcription 3 (STAT3) are a major driver of hepatocellular carcinoma (HCC). Herein, we report a nanointegrative proteolysis-targeting chimera (PROTAC)-based STAT3 degradation strategy that enables efficient chemical reprogramming of HCC-associated CSCs, which potently inhibits CSC growth while evoking anti-HCC immune responses. The PROTAC prodrug was synthesized by conjugating the STAT3 binding domain (inS3) with a thioketal-caged E3 ligase ligand (VL-TK) via an oligo(ethylene glycol) linker (OEG) with tuned length and flexibility and encapsulating it in cRGD-modified cationic liposomes for CSC-targeted delivery while facilitating their lysosomal escape. The PROTAC prodrugs were activated by the upregulated ROS levels in CSCs and efficiently degraded STAT3 for chemical reprogramming, which would not only impair their stemness features but also remodel the immunosuppressive TME into an immunosupportive state to boost anti-HCC immunity. This strategy provides an approach for improving HCC treatment in clinics.

Formation of interfacial molecular film and metal‐trapping function of azacalixarene‐containing copolymers having spherulite‐forming ability

AbstractThe interfacial molecular film behavior of a newly synthesized copolymer having spherulite‐forming ability and consisting of an azacalixarene moiety and a polyethylene glycol linker was investigated. Furthermore, the ability of the azacalixarene ring in the copolymer Langmuir monolayer to trap metals from the subphase was examined. The copolymer formed a monolayer at the air/water interface with high‐density packing. In addition, the corresponding copolymer formed a stable interfacial monolayer whose behavior did not change significantly as the temperature of the subphase changed. The monolayer morphology exhibited a dot‐like crystalline domain. When the subphase contained cadmium ions, the cyclic moiety showed the ability to trap the metal cations. In this case, the distortion angle of the cup‐shaped conformation of the cyclic moiety in the organized molecular film expanded after metal trapping.Highlights LB film of a copolymer containing azacalixarene and PEG units was prepared. The copolymer formed a dense packing in the LB film. Copolymer film formed a surface morphology containing crystalline grains. The copolymer trapped Cd ions in the azacalixarene ring from the subphase. Due to trapping of Cd ions, the distortion angle of the ring widened by 7°.

Significant reduction of activity retention in the kidneys via optimized linker sequences in radiohybrid-based minigastrin analogs

BackgroundWe recently introduced radiohybrid (rh)-based minigastrin analogs e.g., DOTA-rhCCK-18 (DOTA-D-Dap(p-SiFA)-(D-γ-Glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2), that revealed substantially increased activity retention in the tumor. However, one major drawback of these first generation rh-based cholecystokinin-2 receptor (CCK-2R) ligands is their elevated activity levels in the kidneys, especially at later time points (24 h p.i.). Therefore, this study aimed to reduce kidney retention with regard to a therapeutic use via substitution of negatively charged D-glutamic acid moieties by hydrophilic uncharged polyethylene glycol (PEG) linkers of various length ((PEG)4 to (PEG)11). Furthermore, the influence of differently charged silicon-based fluoride acceptor (SiFA)-moieties (p-SiFA: neutral, SiFA-ipa: negatively charged, and SiFAlin: positively charged) on in vitro properties of minigastrin analogs was evaluated. Out of all compounds evaluated in vitro, the two most promising minigastrin analogs were further investigated in vivo.ResultsCCK-2R affinity of most compounds evaluated was found to be in a range of 8–20 nM (by means of apparent IC50), while ligands containing a SiFA-ipa moiety displayed elevated IC50 values. Lipophilicity was noticeably lower for compounds containing a D-γ-glutamate (D-γ-Glu) moiety next to the D-Dap(SiFA) unit as compared to their counterparts lacking the additional negative charge. Within this study, combining the most favorable CCK-2R affinity and lipophilicity, [177/natLu]Lu-DOTA-rhCCK-70 (DOTA-D-Dap(p-SiFA)-D-γ-Glu-(PEG)7-D-γ-Glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2; IC50: 12.6 ± 2.0 nM; logD7.4: − 1.67 ± 0.08) and [177/natLu]Lu-DOTA-rhCCK-91 (DOTA-D-Dap(SiFAlin)-D-γ-Glu-(PEG)4-D-γ-Glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2; IC50: 8.6 ± 0.7 nM; logD7.4 = − 1.66 ± 0.07) were further evaluated in vivo. Biodistribution data of both compounds revealed significantly reduced (p < 0.0001) activity accumulation in the kidneys compared to [177Lu]Lu-DOTA-rhCCK-18 at 24 h p.i., leading to enhanced tumor-to-kidney ratios despite lower tumor uptake. However, overall tumor-to-background ratios of the novel compounds were lower than those of [177Lu]Lu-DOTA-rhCCK-18.ConclusionWe could show that the reduction of negative charges within the linker section of radiohybrid-based minigastrin analogs led to decreased activity levels in the kidneys at 24 h p.i., while maintaining a good tumor uptake. Thus, favorable tumor-to-kidney ratios were accomplished in vivo. However, further optimization has to be done in order to improve tumor retention and general biodistribution profile.Graphical abstract

Quantification of thermodynamic effects of carbohydrate multivalency on avidity using synthetic discrete glycooligomers.

A quantitative understanding of thermodynamic effects of avidity in biomolecular interactions is important. Herein, we synthesized discrete glycooligomers and evaluated their interactions with a model protein using isothermal titration calorimetry. The dimeric glycooligomer exhibited higher binding constants compared to the glycomonomer, attributed to the reduced conformational entropy loss through local presentation of multiple carbohydrate units. Conversely, divalent glycoligands with polyethylene glycol linkers, aiming for multivalent binding, showed enhanced interactions through increased enthalpy. These findings emphasize the importance of distinguishing between the "local avidity" and the "multipoint avidity".

Impact of synthesis methods on the functionality of antibody-conjugated gold nanoparticles for targeted therapy.

Gold nanoparticles (GNPs) are emerging as promising modular platforms for antibody-based cancer therapeutics. Their unique physiochemical properties enable efficient binding of multiple antibodies upon a single particle, thereby enhancing therapeutic potential. However, the effect of widely used synthesis techniques on the characteristics and functionality of antibody-GNP platforms has yet to be fully understood. Here, we investigated the effect of key synthesis approaches, namely, covalent binding and physical adsorption, on the properties and anti-cancer functionality of antibody-coated GNPs. By carefully manipulating synthesis variables, including antibody mass in reaction and linker compositions, we revealed a direct impact of these synthesis methods on antibody binding efficiency and anti-cancer functionality. We found that covalent binding of antibodies to GNPs generated a platform with increased cancer cell killing functionality as compared to the adsorption approach. Additionally, a higher antibody mass in the synthesis reaction and a higher polyethylene glycol linker ratio upon covalently bound antibody-GNPs led to increased cell death. Our findings emphasize the critical role of synthesis strategies in determining the functionality of targeted GNPs for effective cancer therapy.

E7-modified gelatin microcarriers for efficient expansion and stemness preservation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess self-renewal ability, multi-differentiation potential and low immunogenicity, thus serving as an ideal choice for cell therapies. Ex-vivo expansion systems that have been developed to meet clinical demands are faced with two crucial barriers, limited quantity and stemness loss of expanded cells. Hence, it is crucial and feasible to construct microcarriers that can show high and specific affinity to MSCs, and support highly efficient cell expansion with minimal stemness loss. In this study, EPLQLKM (E7) peptides were modified onto gelatin microcarriers by poly (ethylene glycol) (PEG) linkers, which showed great antifouling ability against xenogenic components. The rat bone marrow-derived mesenchymal stem cells (rBMSCs) harvested from the E7-modified gelatin microcarriers achieved better cell attachment, stemness maintenance, viability, and multilineage differentiation potentials, especially those with a higher E7 density. Attributing to the promotion for cell adhesion, E7 functionalization increased the expansion efficiency of rBMSCs with improved quantity and quality simultaneously, thereby providing a novel strategy for scalable expansion to optimize the clinical performance of MSCs.

Solid-State Dewetting of Thin Au Films for Surface Functionalization of Biomedical Implants.

Biomaterial-centered infections of orthopedic implants remain a significant burden in the healthcare system due to sedentary lifestyles and an aging population. One approach to combat infections and improve implant osteointegration is functionalizing the implant surface with anti-infective and osteoinductive agents. In this framework, Au nanoparticles are produced on the surface of Ti-6Al-4V medical alloy by solid-state dewetting of 5 nm Au film and used as the substrate for the conjugation of a model antibiotic vancomycin via a mono-thiolated poly(ethylene glycol) linker. Produced Au nanoparticles on Ti-6Al-4V surface are equiaxed with a mean diameter 19.8 ± 7.2 nm, which is shown by high-resolution scanning electron microscopy and atomic force microscopy. The conjugation of the antibiotic vancomycin, 18.8 ± 1.3 nm-thick film, is confirmed by high resolution-scanning transmission electron microscopy and X-ray photoelectron spectroscopy. Overall, showing a link between the solid-state dewetting process and surface functionalization, we demonstrate a novel, simple, and versatile method for functionalization of implant surfaces.

Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors

PurposeThe lead-203 (203Pb)/lead-212 (212Pb) elementally identical radionuclide pair has gained significant interest in the field of image-guided targeted alpha-particle therapy for cancer. Emerging evidence suggests that 212Pb-labeled peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2) may provide improved effectiveness compared to beta-particle-based therapies for neuroendocrine tumors (NETs). This study aims to improve the performance of SSTR2-targeted radionuclide imaging and therapy through structural modifications to Tyr3-octreotide (TOC)-based radiopharmaceuticals.MethodsNew SSTR2-targeted peptides were designed and synthesized with the goal of optimizing the incorporation of Pb isotopes through the use of a modified cyclization technique; the introduction of a Pb-specific chelator (PSC); and the insertion of polyethylene glycol (PEG) linkers. The binding affinity of the peptides and the cellular uptake of 203Pb-labeled peptides were evaluated using pancreatic AR42J (SSTR2+) tumor cells and the biodistribution and imaging of the 203Pb-labeled peptides were assessed in an AR42J tumor xenograft mouse model. A lead peptide was identified (i.e., PSC-PEG2-TOC), which was then further evaluated for efficacy in 212Pb therapy studies.ResultsThe lead radiopeptide drug conjugate (RPDC) — [203Pb]Pb-PSC-PEG2-TOC — significantly improved the tumor-targeting properties, including receptor binding and tumor accumulation and retention as compared to [203Pb]Pb-DOTA0-Tyr3-octreotide (DOTATOC). Additionally, the modified RPDC exhibited faster renal clearance than the DOTATOC counterpart. These advantageous characteristics of [212Pb]Pb-PSC-PEG2-TOC resulted in a dose-dependent therapeutic effect with minimal signs of toxicity in the AR42J xenograft model. Fractionated administrations of 3.7 MBq [212Pb]Pb-PSC-PEG2-TOC over three doses further improved anti-tumor effectiveness, resulting in 80% survival (70% complete response) over 120 days in the mouse model.ConclusionStructural modifications to chelator and linker compositions improved tumor targeting and pharmacokinetics (PK) of 203/212Pb peptide-based radiopharmaceuticals for NET theranostics. These findings suggest that PSC-PEG2-TOC is a promising candidate for Pb-based targeted radionuclide therapy for NETs and other types of cancers that express SSTR2.

SYNTHESIS OF A POLYFUNCTIONAL DENDRON BASED ON GALLIC ACID USING THE AZIDE-ALKYNE CYCLOADDITION REACTION

By stepwise modification of gallic acid using an azide-alkyne cycloaddition reaction, a first-generation triazole-containing dendron with hydroxypropyltriazole groups and a tetraethylene glycol linker was obtained for the first time. The structure of all intermediate compounds has been proven by modern physical methods. It has been established that the use of bromomethylene derivatives of gallic acid in the synthesis of triazole-containing dendrons results in the formation of by-products of alkylation of the bases used in the reaction (triethylamine and diisopropylethylamine) due to the high mobility of the bromine atom in the benzyl position.

Synthesis of Cross-Chain Bridging Cryptands and Induction of Molecular Chirality.

In this study, we synthesized two cryptands featuring entangled tri- and tetra(ethylene glycol) linkers. The cryptand bearing short linkers was chiral without any asymmetric carbon atoms. After chiral high-performance liquid chromatography was used to separate the enantiomers, the absolute configuration of each cryptand was determined through single-crystal X-ray and circular dichroism analyses. The racemization of the cryptand possessing long linkers proceeded at room temperature.

Preparation of Protein A Membrane Adsorbers Using Strain-Promoted, Copper-Free Dibenzocyclooctyne (DBCO)-Azide Click Chemistry.

Protein A chromatography is the preferred unit operation for purifying Fc-based proteins. Convective chromatography technologies, like membrane adsorbers, can perform the purification rapidly and improve throughput dramatically. While the literature reports the preparation of Protein A membrane adsorbers utilizing traditional coupling chemistries that target lysine or thiol groups on the Protein A ligand, this study demonstrates a new approach utilizing copper-free dibenzocyclooctyne (DBCO)-azide click chemistry. The synthetic pathway consists of three main steps: bioconjugation of Protein A with a DBCO-polyethylene glycol (PEG) linker, preparation of an azide-functionalized membrane surface, and click reaction of DBCO-Protein A onto the membrane surface. Using polyclonal human immunoglobulins (hIgG) as the target molecule, Protein A membranes prepared by this synthetic pathway showed a flowrate-independent dynamic binding capacity of ~10 mg/mL membrane at 10% breakthrough. Fitting of static binding capacity measurements to the Langmuir adsorption isotherm showed a maximum binding (qmax) of 27.48 ± 1.31 mg/mL and an apparent equilibrium dissociation constant (Kd) of value of 1.72 × 10-1 ± 4.03 × 10-2 mg/mL. This work represents a new application for copper-less click chemistry in the membrane chromatography space and outlines a synthetic pathway that can be followed for immobilization of other ligands.

Influence of Surface Ligand Density and Particle Size on the Penetration of the Blood-Brain Barrier by Porous Silicon Nanoparticles.

Overcoming the blood-brain barrier (BBB) remains a significant challenge with regard to drug delivery to the brain. By incorporating targeting ligands, and by carefully adjusting particle sizes, nanocarriers can be customized to improve drug delivery. Among these targeting ligands, transferrin stands out due to the high expression level of its receptor (i.e., transferrin receptor) on the BBB. Porous silicon nanoparticles (pSiNPs) are a promising drug nanocarrier to the brain due to their biodegradability, biocompatibility, and exceptional drug-loading capacity. However, an in-depth understanding of the optimal nanoparticle size and transferrin surface density, in order to maximize BBB penetration, is still lacking. To address this gap, a diverse library of pSiNPs was synthesized using bifunctional poly(ethylene glycol) linkers with methoxy or/and carboxyl terminal groups. These variations allowed us to explore different transferrin surface densities in addition to particle sizes. The effects of these parameters on the cellular association, uptake, and transcytosis in immortalized human brain microvascular endothelial cells (hCMEC/D3) were investigated using multiple in vitro systems of increasing degrees of complexity. These systems included the following: a 2D cell culture, a static Transwell model, and a dynamic BBB-on-a-chip model. Our results revealed the significant impact of both the ligand surface density and size of pSiNPs on their ability to penetrate the BBB, wherein intermediate-level transferrin densities and smaller pSiNPs exhibited the highest BBB transportation efficiency in vitro. Moreover, notable discrepancies emerged between the tested in vitro assays, further emphasizing the necessity of using more physiologically relevant assays, such as a microfluidic BBB-on-a-chip model, for nanocarrier testing and evaluation.

Adsorption of flexible linker for siRNA on carbon nanotube using pyrene anchor: Molecular dynamics simulation

Carbon nanotubes are considered an effective nanoplatform for drug delivery, including therapeutic nucleic acids such as small interfering RNAs (siRNAs), which are used in cancer therapy. In this work, a noncovalent immobilization of a single-stranded oligonucleotide (with 17 nucleotides in length) on the single-walled carbon nanotube (SWNT) surface using a pyrene molecule as an anchor is simulated by molecular dynamics method. This oligonucleotide design supposes the following binding with siRNA ends to provide its keeping near the nanotube surface. In the model, the pyrene molecule is covalently conjugated to the oligonucleotide through an hexaethylene glycol oligomer (EG)6 and attached to the SWNT’s surface by means of π-π stacking interaction. Structures of the complex and the binding energy of pyrene in the complex with SWNT are determined. Two possible orientations of the oligonucleotide arrangement relative to SWNT were considered: mutually perpendicular orientation and arrangement of an oligonucleotide along the nanotube. In both cases, the pyrene-terminated flexible polyethylene glycol linker plays an important role in keeping the oligonucleotide near the nanotube surface and provides sufficiently rapid adsorption of the biopolymer on SWNT, which is important for the creation of new drug delivery systems into the cell and for biosensor design.