Highly aminated iron oxide nanoworms for simultaneous manufacturing and labeling of chimeric antigen receptor T cells.

Cell based therapies including chimeric antigen receptor (CAR) T cells are promising for treating leukemias and solid cancers. At the same time, there is interest in enhancing the functionality of these cells via surface decoration with nanoparticles (backpacking). Magnetic nanoparticle cell labeling is of particular interest due to opportunities for magnetic separation, in vivo manipulation, drug delivery and magnetic resonance imaging (MRI). While modification of T cells with magnetic nanoparticles (MNPs) was explored before, we questioned whether MNPs are compatible with CAR-T cells when introduced during the manufacturing process. We chose highly aminated 120 nm crosslinked iron oxide nanoworms (CLIO NWs, ~36,000 amines per NW) that could efficiently label different adherent cell lines and we used CD123 CAR-T cells as the labeling model. The CD123 CAR-T cells were produced in the presence of CLIO NWs, CLIO NWs plus protamine sulfate (PS), or PS only. The transduction efficiency of lentiviral CD123 CAR with only NWs was ~23% lower than NW+PS and PS groups (~33% and 35%, respectively). The cell viability from these three transduction conditions was not reduced within CAR-T cell groups, though lower compared to non-transduced T cells (mock T). Use of CLIO NWs instead of, or together with cationic protamine sulfate for enhancement of lentiviral transduction resulted in comparable levels of CAR expression and viability but decreased the proportion of CD8+ cells and increased the proportion of CD4+ cells. CD123 CAR-T transduced in the presence of CLIO NWs, CLIO NWs plus PS, or PS only, showed similar level of cytotoxicity against leukemic cell lines. Furthermore, fluorescence microscopy imaging demonstrated that CD123 CAR-T cells labeled with CLIO NW formed rosettes with CD123+ leukemic cells as the non-labeled CAR-T cells, indicating that the CAR-T targeting to tumor cells has maintained after CLIO NW labeling. The in vivo trafficking of the NW labeled CAR-T cells showed the accumulation of CAR-T labeled with NWs primarily in the bone marrow and spleen. CAR-T cells can be magnetically labeled during their production while maintaining functionality using the positively charged iron oxide NWs, which enable the in vivo biodistribution and tracking of CAR-T cells.

Complement Inhibitors Block Complement C3 Opsonization and Improve Targeting Selectivity of Nanoparticles in Blood.

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibody-targeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

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.

Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II.

Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.

Variability of Complement Response toward Preclinical and Clinical Nanocarriers in the General Population.

Opsonization (coating) of nanoparticles with complement C3 component is an important mechanism that triggers immune clearance and downstream anaphylactic and proinflammatory responses. The variability of complement C3 binding to nanoparticles in the general population has not been studied. We examined complement C3 binding to dextran superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoworms, SPIO NWs, 58 and 110 nm) and clinically approved nanoparticles (carboxymethyl dextran iron oxide ferumoxytol (Feraheme, 28 nm), highly PEGylated liposomal doxorubicin (LipoDox, 88 nm), and minimally PEGylated liposomal irinotecan (Onivyde, 120 nm)) in sera from healthy human individuals. SPIO NWs had the highest variation in C3 binding (n = 47) between subjects, with a 15-30 fold range in levels of C3. LipoDox (n = 12) and Feraheme (n = 18) had the lowest levels of variation between subjects (an approximately 1.5-fold range), whereas Onivyde (n = 18) had intermediate between-subject variation (2-fold range). There was no statistical difference between males and females and no correlation with age. There was a significant correlation in complement response between small and large SPIO NWs, which are similar structurally and chemically, but the correlations between SPIO NWs and other types of nanoparticles, and between LipoDox and Onivyde, were not significant. The calculated average number of C3 molecules bound per nanoparticle correlated with the hydrodynamic diameter but was decreased in LipoDox, likely due to the PEG coating. The conclusions of this study are (1) all nanoparticles show variability of C3 opsonization in the general population; (2) an individual's response toward one nanoparticle cannot be reliably predicted based on another nanoparticle; and (3) the average number of C3 molecules per nanoparticle depends on size and surface coating. These results provide new strategies to improve nanomedicine safety.

Radio frequency-mediated local thermotherapy for destruction of pancreatic tumors using Ni-Au core-shell nanowires.

We present a novel method of radio frequency (RF)-mediated thermotherapy in tumors by remotely heating nickel (Ni)-gold (Au) core-shell nanowires (CSNWs). Ectopic pancreatic tumors were developed in nude mice to evaluate the thermotherapeutic effects on tumor progression. Tumor ablation was produced by RF-mediated thermotherapy via activation of the paramagnetic properties of the Ni-Au CSNWs. Histopathology demonstrated that heat generated by RF irradiation caused significant cellular death with pyknotic nuclei and nuclear fragmentation dispersed throughout the tumors. These preliminary results suggest that thermotherapy ablation induced via RF activation of nanowires provides a potential alternative therapy for cancer treatment.

Nanoscale structure-activity relationships, mode of action, and biocompatibility of gold nanoparticle antibiotics.

The emergence of resistance to multiple antimicrobial agents by pathogenic bacteria has become a significant global public health threat. Multi-drug-resistant (MDR) Gram-negative bacteria have become particularly problematic, as no new classes of small-molecule antibiotics for Gram-negative bacteria have emerged in over two decades. We have developed a combinatorial screening process for identifying mixed ligand monolayer/gold nanoparticle conjugates (2.4 nm diameter) with antibiotic activity. The method previously led to the discovery of several conjugates with potent activity against the Gram-negative bacterium Escherichia coli. Here we show that these conjugates are also active against MDR E. coli and MDR Klebsiella pneumoniae. Moreover, we have shown that resistance to these nanoparticles develops significantly more slowly than to a commercial small-molecule drug. These results, combined with their relatively low toxicity to mammalian cells and biocompatibility in vivo, suggest that gold nanoparticles may be viable new candidates for the treatment of MDR Gram-negative bacterial infections.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Inhibition of acute complement responses towards bolus-injected nanoparticles using targeted short-circulating regulatory proteins.

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation.

KDEL peptide gold nanoconstructs: promising nanoplatforms for drug delivery.

Gold nanoparticles (AuNPs) have been widely investigated as potential nanocarriers for drug delivery. In the present study, AuNPs were conjugated to a peptide that has a C-terminal Lys-Asp-Glu-Leu (KDEL) motif. In a pulse-chase study, time-course sampling revealed that AuNP-delivered KDEL peptides were rapidly localized to the endoplasmic reticulum (ER) in 5 to 15 min, and after 1h the majority of peptides were localized to the ER. Clathrin-coated vesicles and Golgi apparatus were also involved during the intracellular trafficking of KDEL peptide gold (AuNP-KDEL) nanoconstructs. Furthermore, overexpression of KDEL receptor (KDELR) significantly enhanced KDEL peptide uptake in both free and AuNP-conjugated forms. These data indicate that the AuNP-KDEL nanoconstructs are internalized via a clathrin-mediated pathway and trafficked to the ER via a retrograde transport pathway, bypassing the lysosomal degradation pathway. Thus, this novel approach to development of nanoconstruct-based drug delivery has the potential to evade intracellular degradation, enhancing drug efficacy. FROM THE CLINICAL EDITOR: In this study, gold nanoparticles were conjugated to a peptide with KDEL motif, resulting in internalization via a clathrin-mediated pathway and trafficking to the ER via retrograde transport meanwhile bypassing the lysosomal degradation pathway. This method results in a potential evasion of intracellular degradation, and enhanced drug efficacy.

Gold nanoparticles: the importance of physiological principles to devise strategies for targeted drug delivery.

Nanotechnology and its promise for clinical translation to targeted drug delivery with limited accompanying toxicity provide exciting research opportunities that demand multidisciplinary approaches. To make rapid progress in the design of nano-platforms for drug delivery and toward their use in the clinic, basic and mechanistic studies must first be tested in vitro and then progress to in vivo studies in animal models, incorporating an understanding of body functioning. Recently, gold nanoparticles (Au NPs) have gained much attention as model drug delivery platforms because of their advantageous surface characteristics that allow easy functionalization with chemical and biological molecules and also due to their apparently low toxicity. In this study we review recent in vitro and in vivo research progress with Au NPs as drug delivery platforms and suggest experimental strategies for future studies for efficacious, targeted delivery.

Gold-peptide nanoconjugate cellular uptake is modulated by serum proteins.

Gold nanoparticles (Au NPs, 20 nm) were conjugated with two different cysteine-terminated peptides. Radio-ligand binding studies were conducted to characterize Au NP-peptide binding, suggesting both covalent and noncovalent interactions. The interactions of serum proteins with Au NP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugates (15 minutes). The cellular uptake of free peptides and nanoconjugates into mouse myogenic (Sol8) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence signals compared to those in the presence of serum (P < 0.05), suggesting that serum proteins inhibit Au NP-mediated peptide delivery. The cellular uptake of nanoconjugates was also confirmed using transmission electron microscopy. These data suggest that Au NP-peptide nanoconjugates are a useful platform for intracellular delivery of therapeutics. However, a deeper understanding of the mechanisms regulating their uptake and intracellular trafficking is needed.

Surface Modification of Erythrocytes with Lipid Anchors: Structure-Activity Relationship for Optimal Membrane Incorporation, in vivo Retention, and Immunocompatibility.

Red blood cells (RBCs) are natural carriers for sustained drug delivery, imaging, and in vivo sensing. One of the popular approaches to functionalize RBCs is through lipophilic anchors, but the structural requirements for anchor stability and in vivo longevity remain to be investigated. Using fluorescent lipids with the same cyanine 3 (Cy3) headgroup but different lipid chain and linker, the labeling efficiency of RBCs and in vivo stability are investigated. Short-chain derivatives exhibited better insertion efficiency, and mouse RBCs are better labeled than human RBCs. Short-chain derivatives demonstrate low retention in vivo. Derivatives with ester bonds are especially unstable, due to removal and degradation. On the other hand, long-chain, covalently linked derivatives show remarkably long retention and stability (over 80 days half life in the membrane). The clearance organs are liver and spleen with evidence of lipid transfer to the liver sinusoidal endothelium. Notably, RBCs modified with PEGylated lipid show decreased macrophage uptake. Some of the derivatives promote binding of antibodies in human plasma and mouse sera and modest increase in complement deposition and hemolysis, but these do not correlate with in vivo stability of RBCs. Ultra-stable anchors can enable functionalization of RBCs for drug delivery, imaging, and sensing.

PEGylated Liposomes Accumulate in the Areas Relevant to Skin Toxicities via Passive Extravasation across "Leaky" Endothelium.

PEGylated liposome is the cornerstone platform for modern drug delivery. Unfortunately, as exemplified by PEGylated liposomal doxorubicin (aka Doxil), altered doxorubicin pharmacokinetics causes off-target accumulation in the skin, including the palms and feet, leading to severe dose-limiting toxicity. In addition to Doxil, other nanoparticles and PEGylated liposomes exhibit significant deposition in the skin, but mechanisms of accumulation are poorly understood. Using ex vivo imaging and ex vivo confocal microscopy, we show that PEGylated liposomes in mice accumulate predominantly in the areas subject to mechanical stress/pressure. Blood vessels in foot skin appear to be especially leaky, exhibiting burst-like extravasations. Using high-resolution confocal microscopy and liposomes labeled with different dyes in the membrane and/or interior, two modes of extravasation were observed: (1) as intact liposomes; (2) as separated liposomal components. On the other hand, stable cross-linked iron oxide nanoworms extravasated only as intact nanoparticles. There was no colocalization between liposomes and exosomal marker CD81, excluding the role of exocytosis. Also, in situ perfusion of formalin-fixed foot skin with labeled liposomes revealed that the extravasation is mediated by passive, energy-independent diffusion and not by leukocyte "hitchhiking". These findings improve our understanding of extravasation pathways of nanocarriers in the areas relevant to skin pathologies and could lead to strategies to prevent and treat liposome-induced skin toxicities.

Indocarbocyanine nanoparticles extravasate and distribute better than liposomes in brain tumors.

Glioblastoma (GBM) is the most devastating and aggressive brain tumor in adults. Hidden behind the blood-brain and blood-tumor barriers (BBTB), this invasive type of brain tumor is not readily accessible to nano-sized particles. Here we demonstrate that fluorescent indocarbocyanine lipids (ICLs: DiD, DiI) formulated in PEGylated lipid nanoparticle (PLN) exhibit highly efficient penetration and accumulation in GBM. PLN-formulated ICLs demonstrated more efficient penetration in GBM spheroids and organoids in vitro than liposomal ICLs. Over 82% of the tumor's extravascular area was positive for ICL fluorescence in the PLN group versus 13% in the liposomal group just one hour post-systemic injection in the intracranial GBM model. Forty-eight hours post-injection, PLN-formulated ICLs accumulated in 95% of tumor myeloid-derived suppressor cells and macrophages, 70% of tumor regulatory T cells, 50% of tumor-associated microglia, and 65% of non-immune cells. PLN-formulated ICLs extravasated better than PEGylated liposomal doxorubicin and fluorescent dextran and efficiently accumulated in invasive tumor margins and brain-invading cells. While liposomes were stable in serum in vitro and in vivo, PLNs disassembled before entering tumors, which could explain the differences in their extravasation efficiency. These findings offer an opportunity to improve therapeutic cargo delivery to invasive GBM.

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on "Pseudovirus-like" Nanoparticles.

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naïve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, "pseudovirus-like" nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

Liposomal Extravasation and Accumulation in Tumors as Studied by Fluorescence Microscopy and Imaging Depend on the Fluorescent Label.

Tumor trafficking of liposomes is routinely monitored via fluorescence microscopy and imaging. To investigate whether an accumulation of liposomes depends on the type of fluorescent label, we prepared PEGylated liposomes dual-labeled with indocarbocyanine lipids (ICLs: DiD or DiI) and fluorescent phospholipids (FPLs: Cy3-DSPE or Cy5-DSPE) with similar cyanine headgroups but different spectra. Using ex vivo confocal microscopy and imaging, we compared tumor extravasation and accumulation of ICLs and FPLs. After systemic injection in a syngeneic mouse model of 4T1 breast cancer, ICLs and FPLs initially colocalized in tumor blood vessels and perivascular space. At later time points, ICLs spread over a significantly larger tumor area and accumulated in tumor macrophages, whereas FPLs were mostly restricted to the vasculature with limited extravascular signal. This phenomenon was independent of liposomal composition and ICL/FPL type and was also observed in syngeneic intracranial GL261 glioma and LY2 head and neck cancer models. The dual-labeled liposomes were stable in plasma and delivered both dyes to tumors at early time points. Notably, while the level of ICLs increased over time, FPLs gradually disappeared from tumors and other organs in vivo, likely due to degradation of the phospholipid. These findings demonstrate that trafficking and stability of the label is of critical importance when assessing extravasation and accumulation of nanocarriers in tumors and other organs by fluorescence microscopy and imaging.

Targeted Intracellular Delivery of Trastuzumab Using Designer Phage Lambda Nanoparticles Alters Cellular Programs in Human Breast Cancer Cells.

| Several diseases exhibit a high degree of heterogeneity and diverse reprogramming of cellular pathways. To address this complexity, additional strategies and technologies must be developed to define their scope and variability with the goal of improving current treatments. Nanomedicines derived from viruses are modular systems that can be easily adapted for combinatorial approaches, including imaging, biomarker targeting, and intracellular delivery of therapeutics. Here, we describe a "designer nanoparticle" system that can be rapidly engineered in a tunable and defined manner. Phage-like particles (PLPs) derived from bacteriophage lambda possess physiochemical properties compatible with pharmaceutical standards, and in vitro particle tracking and cell targeting are accomplished by simultaneous display of fluorescein-5-maleimide (F5M) and trastuzumab (Trz), respectively (Trz-PLPs). Trz-PLPs bind to the oncogenically active human epidermal growth factor receptor 2 (HER2) and are internalized by breast cancer cells of the HER2 overexpression subtype, but not by those lacking the HER2 amplification. Compared to treatment with Trz, robust internalization of Trz-PLPs results in higher intracellular concentrations of Trz, prolonged inhibition of cell growth, and modulated regulation of cellular programs associated with HER2 signaling, proliferation, metabolism, and protein synthesis. Given the implications to cancer pathogenesis and that dysregulated signaling and metabolism can lead to drug resistance and cancer cell survival, the present study identifies metabolic and proteomic liabilities that could be exploited by the PLP platform to enhance therapeutic efficacy. The lambda PLP system is robust and rapidly modifiable, which offers a platform that can be easily "tuned" for broad utility and tailored functionality.

Complement opsonization of nanoparticles: Differences between humans and preclinical species.

The complement system plays a key role in opsonization and immune clearance of engineered nanoparticles. Understanding the efficiency, inter-subject, and inter-strain differences of complement opsonization in preclinical species can help with translational nanomedicine development and improve our ability to model complement response in humans. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles and a wide range of non-magnetic iron oxide nanoparticle formulations are widely used in magnetic resonance imaging and as clinically approved iron supplements. Previously we found that opsonization of SPIO nanoworms (NW) with the third complement protein (C3) proceeds mostly via the alternative pathway in humans, and via the lectin pathway in mice. Here, we studied the pathway and efficiency of opsonization of 106 nm SPIO NW with C3 in different preclinical species and commonly used laboratory strains. In sera of healthy human donors (n = 6), C3 opsonization proceeded exclusively through the alternative pathway. On the other hand, the C3 opsonization in dogs (6 breeds), rats (4 strains) and mice (5 strains) sera was either partially or completely dependent on the complement Ca2+-sensitive pathways (lectin and/or classical). Specifically, C3 opsonization in sera of Long Evans rat strain, and mouse strains widely used in nanomedicine research (BALB/c, C57BL/6 J, and A/J) was only through the Ca2+-dependent pathways. Dogs and humans had the highest between-subject variability in C3 opsonization levels, while rat and mouse sera showed the lowest between-strain variability. Furthermore, using a panel of SPIO nanoparticles of different sizes and dextran coatings, we found that the level of C3 opsonization (C3 molecules per milligram Fe) in human sera was lower than in animal sera. At the same time, there was a strong predictive value of complement opsonization in dog and rat sera; nanoparticles with higher C3 deposition in animals showed higher deposition in humans, and vice versa. Notably, the opsonization decreased with decreasing size in all sera. The studies highlight the importance of the consideration of species and strains for predicting human complement responses (opsonization) towards nanomedicines.