Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Optimized Enzyme-Linked Immunosorbent Assay for Anti-PEG Antibody Detection in Healthy Donors and Patients Treated with PEGylated Liposomal Doxorubicin.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG5000, as opposed to bovine serum albumin-PEG20000, and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

Perspectives on complement and phagocytic cell responses to nanoparticles: From fundamentals to adverse reactions.

The complement system, professional phagocytes and other cells such as Natural killer cells and mast cells are among the important components of the innate arm of the immune system. These constituents provide an orchestrated array of defences and responses against tissue injury and foreign particles, including nanopharmaceuticals. While interception of nanopharmaceuticals by the immune system is beneficial for immunomodulation and treatment of phagocytic cell disorders, it is imperative to understand the multifaceted mechanisms by which nanopharmaceuticals interacts with the immune system and evaluate the subsequent balance of beneficial versus adverse reactions. An example of the latter is adverse infusion reactions to regulatory-approved nanopharmaceuticals seen in human subjects. Here, we discuss collective opinions and findings from our laboratories in mapping nanoparticle-mediated complement and leucocyte/macrophage responses.

Microneedle-based devices for point-of-care infectious disease diagnostics.

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.

Roadmap on nanomedicine.

Since the launch of the Alliance for Nanotechnology in Cancer by the National Cancer Institute in late 2004, several similar initiatives have been promoted all over the globe with the intention of advancing the diagnosis, treatment and prevention of cancer in the wake of nanoscience and nanotechnology. All this has encouraged scientists with diverse backgrounds to team up with one another, learn from each other, and generate new knowledge at the interface between engineering, physics, chemistry and biomedical sciences. Importantly, this new knowledge has been wisely channeled towards the development of novel diagnostic, imaging and therapeutic nanosystems, many of which are currently at different stages of clinical development. This roadmap collects eight brief articles elaborating on the interaction of nanomedicines with human biology; the biomedical and clinical applications of nanomedicines; and the importance of patient stratification in the development of future nanomedicines. The first article reports on the role of geometry and mechanical properties in nanomedicine rational design; the second articulates on the interaction of nanomedicines with cells of the immune system; and the third deals with exploiting endogenous molecules, such as albumin, to carry therapeutic agents. The second group of articles highlights the successful application of nanomedicines in the treatment of cancer with the optimal delivery of nucleic acids, diabetes with the sustained and controlled release of insulin, stroke by using thrombolytic particles, and atherosclerosis with the development of targeted nanoparticles. Finally, the last contribution comments on how nanomedicine and theranostics could play a pivotal role in the development of personalized medicines. As this roadmap cannot cover the massive extent of development of nanomedicine over the past 15 years, only a few major achievements are highlighted as the field progressively matures from the initial hype to the consolidation phase.

The Interplay Between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses.

In the blood, depending on their physicochemical characteristics, nanoparticles attract a wide range of plasma biomolecules. The majority of blood biomolecules bind nonspecifically to nanoparticles. On the other hand, biomolecules such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclinical and clinical nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.

Determination of Polycation-Mediated Perturbation of Mitochondrial Respiration in Intact Cells by High-Resolution Respirometry (Oxygraph-2k, OROBOROS).

A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.

Combined Fluorimetric Caspase-3/7 Assay and Bradford Protein Determination for Assessment of Polycation-Mediated Cytotoxicity.

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell-death related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of caspase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Mechanisms and Barriers in Cancer Nanomedicine: Addressing Challenges, Looking for Solutions.

Remarkable progress has recently been made in the synthesis and characterization of engineered nanoparticles for imaging and treatment of cancers, resulting in several promising candidates in clinical trials. Despite these advances, clinical applications of nanoparticle-based therapeutic/imaging agents remain limited by biological, immunological, and translational barriers. In order to overcome the existing status quo in drug delivery, there is a need for open and frank discussion in the nanomedicine community on what is needed to make qualitative leaps toward translation. In this Nano Focus, we present the main discussion topics and conclusions from a recent workshop: "Mechanisms and Barriers in Nanomedicine". The focus of this informal meeting was on biological, toxicological, immunological, and translational aspects of nanomedicine and approaches to move the field forward productively. We believe that these topics reflect the most important issues in cancer nanomedicine.

AFM visualization of sub-50nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope.

It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50nm particles resembling polyplexes in morphology compared with control preparations.

Activation of Human Complement System by Dextran-Coated Iron Oxide Nanoparticles Is Not Affected by Dextran/Fe Ratio, Hydroxyl Modifications, and Crosslinking.

While having tremendous potential as therapeutic and imaging tools, the clinical use of engineered nanoparticles has been associated with serious safety concerns. Activation of the complement cascade and the release of proinflammatory factors C3a and C5a may contribute to infusion-related reactions, whereas opsonization with C3 fragments promotes rapid recognition and clearance of nanomaterials by mononuclear phagocytes. We used dextran-coated superparamagnetic iron oxide nanoparticles (SPIO), which are potent activators of the complement system, to study the role of nanoparticle surface chemistry in inciting complement in human serum. Using complement inhibitors and measuring levels of fluid phase markers (sC5b-9, C5a, and Bb), we found that the majority of human complement activation by SPIO is through the alternative pathways (AP). SPIO prepared with high dextran/iron ratio showed some complement activation via calcium-sensitive pathways, but the AP was responsible for the bulk of complement activation and amplification. Activation via the AP required properdin, the positive regulator of the alternative C3bBb convertase. Modification of sugar alcohols of dextran with alkylating, acylating, or crosslinking agents did not overcome complement activation and C3 opsonization. These data demonstrate that human complement activation is independent of dextran modification of SPIO and suggest a crucial role of the AP in immune recognition of nano-assemblies in human serum.

An integrated assessment of morphology, size, and complement activation of the PEGylated liposomal doxorubicin products Doxil®, Caelyx®, DOXOrubicin, and SinaDoxosome.

In order to improve patient's benefit and safety, comprehensive regulatory guidelines on specificities of Non-Biological Complex Drugs (NBCDs), such as doxorubicin-encapsulated liposomes, and their follow-on versions are needed. Here, we compare Doxil® and its European analog Caelyx® with the two follow-on products DOXOrubicin (approved by the US Food and Drug Administration) and SinaDoxosome (produced in Iran) by cryogenic transmission electron microscopy, dynamic light scattering and Nanoparticle Tracking Analysis, and assess their potential in activating the complement system in human sera. We found subtle physicochemical differences between the tested liposomal products and even between the tested batches of Doxil® and Caelyx®. Notably, these included differences in vesicular population aspect ratios and particle number. Among the tested products, only SinaDoxosome, in addition to the presence of unilamellar vesicles with entrapped doxorubicin crystals, contained empty circular disks. Differences were also found in complement responses, which may be related to some morphological differences. This study has demonstrated an integrated biophysical and immunological toolbox for improved analysis and detection of physical differences among vesicular populations that may modulate their clinical performance. Combined, these approaches may help better product selection for infusion to the patients as well as for improved design and characterization of future vesicular NBCDs with enhanced clinical performance and safety.

Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction: basic guidelines.

A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requires interdisciplinary research platforms and techniques for a more profound understanding of biophysical properties of delivery vehicles and their biological performance, including stability, transfection efficacy and possible cytotoxicity. Fluorescent microscopy has proven to be a useful tool for real-time monitoring of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes.

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy.

BACKGROUND: Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells. METHODS: We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated. RESULTS: The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells. CONCLUSIONS: The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells. GENERAL SIGNIFICANCE: Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.

Structural profiling and biological performance of phospholipid-hyaluronan functionalized single-walled carbon nanotubes.

In spite of significant insolubility and toxicity, carbon nanotubes (CNTs) erupt into the biomedical research, and create an increasing interest in the field of nanomedicine. Single-walled CNTs (SWCNTs) are highly hydrophobic and have been shown to be toxic while systemically administrated. Thus, SWCNTs have to be functionalized to render water solubility and biocompatibility. Herein, we introduce a method for functionalizing SWCNT using phospholipids (PL) conjugated to hyaluronan (HA), a hydrophilic glycosaminoglycan, with known receptors on many types of cancer and immune cells. This functionalization allowed for CNT solubilization, endowed the particles with stealth properties evading the immune system, and reduced immune and mitochondrial toxicity both in vitro and in vivo. The CNT-PL-HA internalized into macrophages and showed low cytotoxicity. In addition, CNT-PL-HA did not induce an inflammatory response in macrophages as evidenced by the cytokine profiling and the use of image-based high-content analysis approach in contrast to non-modified CNTs. In addition, systemic administration of CNT-PL-HA into healthy C57BL/6 mice did not alter the total number of leukocytes nor increased liver enzyme release as opposed to CNTs. Taken together, these results suggest an immune protective mechanism by the PL-HA coating that could provide future therapeutic benefit.

Complement monitoring of Pluronic 127 gel and micelles: suppression of copolymer-mediated complement activation by elevated serum levels of HDL, LDL, and apolipoproteins AI and B-100.

Poloxamer 407 is a non-ionic polyethylene oxide (PEO)/polypropylene oxide (PPO) block copolymer, which exhibits reversible thermogelation properties. Poloxamer gel has attracted many applications for controlled release of therapeutic agents as well as in surgical interventions such as controlled vascular occlusion. We show that poloxamer gel can trigger the complement system, which is an integral part of innate immunity and its inadvertent activation can induce clinically significant anaphylaxis. Complement activation by the poloxamer gel is through the alternative pathway, but material transformations from gel to the solution state further incite complement through calcium-sensitive pathways, where a role for C1q and antibodies has been eliminated. Poloxamer addition to plasma/serum (at levels above its critical micelle concentration, cmc) induced formation of large and diffused structures, which may have been responsible for triggering complement. Since poloxamer 407 administration has been reported to cause significant changes in plasma cholesterol and triglyceride levels we further examined the role of lipoproteins in poloxamer-mediated complement activation. Our results show a protective role for elevated serum HDL, LDL and their predominant apolipoproteins (apoAI and apoB-100, respectively) on poloxamer-mediated complement activation. Electron microscopy investigations indicated formation of two distinct populations of new structures on mixing of poloxamer (at concentrations above its cmc) with human LDL, which could have played a significant role in regulating complement activation. These observations are in line with the suggested modulatory role of lipoproteins in host defence and inflammatory processes. A better understanding of block copolymer interaction with lipoproteins/apolipoproteins could improve the immune safety of surgical and therapeutic interventions requiring PEO/PPO block copolymers and may provide new insights for combinatorial design of multifunctional copolymers.

Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents.

Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking capacity of soluble antigen still remain. Here, we address these issues using a novel CAR binding moiety based on the oligoclonal camelid single domain antibodies. A unique set of 13 single domain antibodies were selected from an immunized camel phage library based on their target specificity and binding affinity. A combination of these single domain antibodies was used to generate four tumor associated glycoprotein (TAG-72)-specific CARs harboring an identical antigen binding site, but with different signaling and spacer domains. Although all four CARs were functionally active against the TAG-72 expressing tumor cells, the combination of CD3ζ, OX40, CD28 as well as the CH3-CH2-hinge-hinge domains most efficiently triggered T cell activation. Importantly, CAR mediated functions were not blocked by the soluble TAG-72 antigen at a supraphysiological concentration. Our approach may have the potential to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs.

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.