Targeted nanoparticles have been extensively explored for their ability to deliver their payload to a selective cell population while reducing off-target side effects. The design of actively targeted nanoparticles requires the grafting of a ligand that specifically binds to a highly expressed receptor on the surface of the targeted cell population. Optimizing the interactions between the targeting ligand and the receptor can maximize the cellular uptake of the nanoparticles and subsequently improve their activity. Here, we evaluated how the density and presentation of the targeting ligands dictate the cellular uptake of nanoparticles. To do so, we used a DNA-scaffolded PLGA nanoparticle system to achieve efficient and tunable ligand conjugation. A prostate-specific membrane antigen (PSMA) expressing a prostate cancer cell line was used as a model. The density and presentation of PSMA targeting ligand ACUPA were precisely tuned on the DNA-scaffolded nanoparticle surface, and their impact on cellular uptake was evaluated. It was found that matching the ligand density with the cell receptor density achieved the maximum cellular uptake and specificity. Furthermore, DNA hybridization-mediated targeting chain rigidity of the DNA-scaffolded nanoparticle offered ∼3 times higher cellular uptake compared to the ACUPA-terminated PLGA nanoparticle. Our findings also indicated a ∼ 3.7-fold reduction in the cellular uptake for the DNA hybridization of the non-targeting chain. We showed that nanoparticle uptake is energy-dependent and follows a clathrin-mediated pathway. Finally, we validated the preferential tumor targeting of the nanoparticles in a bilateral tumor xenograft model. Our results provide a rational guideline for designing actively targeted nanoparticles and highlight the application of DNA-scaffolded nanoparticles as an efficient active targeting platform.

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.

Stepwise change between low and high salt concentration buffers of the same pH results in pH transition, the length of which was demonstrated to be proportional to the quantity of ion-exchange groups present on the matrix. In this work, we analyzed the effect of the ligand type, density, and buffer concentration on the pH transition shape for typical ion-exchange groups (QA, DEAE, SO3, and COOH) and ligands acting as metal-chelators, such as IDA, TAEA, and EDA. It was demonstrated that pH transition can occur either as a chromatographic or flat-top peak. pH transition peaks were evaluated by their length, height, and peak center parameters. While no parameter can describe the ligand density accurately with a single linear correlation for both peak types, all parameters can be used for the description of one peak type. Peak length and height exhibited the same accuracy, while their sensitivity depended on the pH transition shape: length being more sensitive for the flat-top peaks, while height for the chromatographic peaks. pH height can be obtained faster, at lower elution volume, and seems to be more suitable for the determination of low amounts of ligand, when typically chromatographic peak type pH transitions occur.

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high-capacity polymer-grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD-grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD-grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high-capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300-350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD-grafted protein A gels than non-grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.

Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion-exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps. Ligand density is known to be one of the main lot-to-lot variations, affecting protein adsorption and separation performance. The use of a model-based approach can be an effective tool for comprehending the impact of parameter variations (e.g., ligand density) and their influence on the process. The objective of this work was to apply mechanistic modeling to gain a deeper understanding of the influence of ligand density variations in anion exchange chromatography. To achieve this, 13 prototype resins having the same support as the strong anion exchange resin Fractogel® EMD TMAE (M), but differing in ligand density, were analyzed. Linear salt gradient elution experiments were performed to observe the elution behavior of a monoclonal antibody and bovine serum albumin. A proposed isotherm model for ion exchange chromatography, describing the dependence of ligand density variations on protein retention, was successfully applied.

The newest generation of drug delivery systems (DDSs) exploits ligands to mediate specific targeting of cells and/or tissues. However, studies investigating the link between ligand density and nanoparticle (NP) uptake are limited to a small number of ligand-receptor systems. C-type lectin-like molecule-1 (CLL1) is uniquely expressed on myeloid cells, which enables the development of receptors specifically targeting treat various diseases. This study aims to investigate how NPs with different CLL1 targeting peptide density impact cellular uptake. To this end, poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs are functionalized with cyclized CLL1 binding peptides (cCBP) ranging from 240 ± 12 to 31 000 ± 940 peptides per NP. Unexpectedly, the percentage of cells with internalized NPs is decreased for all cCBP-NP designs regardless of ligand density compared to unmodified NPs. Internalization through CLL1 receptor-mediated processes is further investigated without confounding the effects of NP size and surface charge. Interestingly, high density cCBP-NPs (>7000 cCBP per NP) uptake is dominated by CLL1 receptor-mediated processes while low density cCBP-NPs (≈200 cCBP per NP) and untargeted NP occurred through non-specific clathrin and caveolin-mediated endocytosis. Altogether, these studies show that ligand density and uptake mechanism should be carefully investigated for specific ligand-receptor systems for the design of targeted DDSs to achieve effective drug delivery.

With significantly decreased light scattering and tissue autofluorescence, fluorescence imaging in the second near infrared (NIR-II, 1000-1700 nm) region has been heavily explored in biomedical field recently. Silver sulfide quantum dots (Ag2S QDs) with unique optical properties were one of the most classic NIR-II imaging probes. However, the Ag2S QDs for in vivo purpose were mainly obtain by oil phase-based high-temperature route at present. Here, we proposed a mild aqueous route to prepare NIR-II emissive Ag2S QDs for in vivo tumor imaging. Original Ag2S QDs was obtained by mixing sodium sulfide and silver nitrate in a thiol-terminated polyethylene glycol (mPEG-SH) solution. Treating the original Ag2S QDs with extra mPEG-SH ligands produced highly PEGyalted Ag2S QDs. These re-PEGylated Ag2S QDs exhibited much better blood circulation and tumor accumulation in vivo comparing with the original ones, which can serve as excellent tumor imaging probes. The whole-body blood vessel imaging of living mice was achieved with high resolution, the bio-distribution of these QDs were studied by NIR-II imaging as well. This work also highlighted the importance of ligand density for tumor targeting.

Monoclonal antibodies are key molecules in medicine and pharmaceuticals. A potentially crucial drawback for faster advances in research here is their high price due to the extremely expensive antibody purification process, particularly the affinity capture step. Affinity chromatography materials have to demonstrate the high binding capacity and recovery efficiency as well as superior chemical and mechanical stability. Low-cost materials and robust, faster processes would reduce costs and enhance industrial immunoglobulin purification. Therefore, exploring the use of alternative materials is necessary. In this context, we conduct the first comparison of the performance of magnetic nanoparticles with commercially available chromatography resins and magnetic microparticles with regard to immobilizing Protein G ligands and recovering immunoglobulin G (IgG). Simultaneously, we demonstrate the suitability of bare as well as silica-coated and epoxy-functionalized magnetite nanoparticles for this purpose. All materials applied have a similar specific surface area but differ in the nature of their matrix and surface accessibility. The nanoparticles are present as micrometer agglomerates in solution. The highest Protein G density can be observed on the nanoparticles. IgG adsorbs as a multilayer on all materials investigated. However, the recovery of IgG after washing indicates a remaining monolayer, which points to the specificity of the IgG binding to the immobilized Protein G. One important finding is the impact of the ligand-binding stoichiometry (Protein G surface coverage) on IgG recovery, reusability, and the ability to withstand long-term sanitization. Differences in the materials\' performances are attributed to mass transfer limitations and steric hindrance. These results demonstrate that nanoparticles represent a promising material for the economical and efficient immobilization of proteins and the affinity purification of antibodies, promoting innovation in downstream processing.

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell-particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin-II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand-free 2 kDa short PEG chains show a superior accumulation in angiotensin-II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1-3 × 10-9 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.

In the study of multivalent interactions at interfaces, as occur for example at cell membranes, the density of the ligands or receptors displayed at the interface plays a pivotal role, affecting both the overall binding affinities and the valencies involved in the interactions. In order to control the ligand density at the interface, several approaches have been developed, and they concern the functionalization of a wide range of materials. Here, different methods employed in the modification of surfaces with controlled densities of ligands are being reviewed. Examples of such methods encompass the formation of self-assembled monolayers (SAMs), supported lipid bilayers (SLBs) and polymeric layers on surfaces. Particular emphasis is given to the methods employed in the study of different types of multivalent biological interactions occurring at the functionalized surfaces and their working principles.