Allan E. David

Polyethylene glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting.

While successful magnetic tumor targeting of iron oxide nanoparticles has been achieved in a number of models, the rapid blood clearance of magnetically suitable particles by the reticuloendothelial system (RES) limits their availability for targeting. This work aimed to develop a long-circulating magnetic iron oxide nanoparticle (MNP) platform capable of sustained tumor exposure via the circulation and, thus, potentially enhanced magnetic tumor targeting. Aminated, cross-linked starch (DN) and aminosilane (A) coated MNPs were successfully modified with 5 kDa (A5, D5) or 20 kDa (A20, D20) polyethylene glycol (PEG) chains using simple N-Hydroxysuccinimide (NHS) chemistry and characterized. Identical PEG-weight analogues between platforms (A5 & D5, A20 & D20) were similar in size (140-190 nm) and relative PEG labeling (1.5% of surface amines - A5/D5, 0.4% - A20/D20), with all PEG-MNPs possessing magnetization properties suitable for magnetic targeting. Candidate PEG-MNPs were studied in RES simulations in vitro to predict long-circulating character. D5 and D20 performed best showing sustained size stability in cell culture medium at 37 °C and 7 (D20) to 10 (D5) fold less uptake in RAW264.7 macrophages when compared to previously targeted, unmodified starch MNPs (D). Observations in vitro were validated in vivo, with D5 (7.29 h) and D20 (11.75 h) showing much longer half-lives than D (0.12 h). Improved plasma stability enhanced tumor MNP exposure 100 (D5) to 150 (D20) fold as measured by plasma AUC(0-∞). Sustained tumor exposure over 24 h was visually confirmed in a 9L-glioma rat model (12 mg Fe/kg) using magnetic resonance imaging (MRI). Findings indicate that a polyethylene glycol modified, cross-linked starch-coated MNP is a promising platform for enhanced magnetic tumor targeting, warranting further study in tumor models.

Magnetic brain tumor targeting and biodistribution of long-circulating PEG-modified, cross-linked starch coated iron oxide nanoparticles

Magnetic iron oxide nanoparticles (MNPs) have been studied to circumvent the limitations of status-quo brain tumor therapy and can be targeted by applying an external magnetic field to lesions. To address the pharmacokinetic shortcomings of MNPs that can limit targeting efficiency, we recently reported a long-circulating polyethylene glycol modified, cross-linked starch MNP (PEG-MNP) suitable for magnetic targeting. Using a rat model, this work explores the biodistribution patterns of PEG-MNPs in organs of elimination (liver, spleen, lung, and kidney) and shows proof-of-concept that enhanced magnetic brain tumor targeting can be achieved due to the relatively long circulation lifetime of the nanoparticles. Reductions in liver (∼12-fold) and spleen (∼2.5-fold) PEG-MNP concentrations at 1h compared to parent starch-coated MNPs (D) confirm plasma pharmacokinetics observed previously. While liver concentrations of PEG-MNPs remained considerably lower than those observed for D at 1h through 60 h, spleen values continue to increase and are markedly higher at later time points--a trend also observed with histology. Limited to no distribution of PEG-MNPs was visualized in lung or kidney throughout the 60 h course evaluated. Enhanced, selective magnetic brain tumor targeting (t = 1 h) of PEG-MNPs (12 mg Fe/kg) was confirmed in 9L-glioma tumors, with up to 1.0% injected dose/g tissue nanoparticle delivery achieved--a 15-fold improvement over targeted D (0.07% injected dose/g tissue). MRI and histological analyses visually confirmed enhanced targeting and also suggest a limited contribution of passive mechanisms to tissue retention of nanoparticles. Our results are exciting and justify both further development of PEG-MNP as a drug delivery platform and concurrent optimization of the magnetic brain tumor targeting strategy utilized.

Brain tumor targeting of magnetic nanoparticles for potential drug delivery: effect of administration route and magnetic field topography.

Our previous studies demonstrated feasibility of magnetically-mediated retention of iron oxide nanoparticles in brain tumors after intravascular administration. The purpose of this study was to elucidate strategies for further improvement of this promising approach. In particular, we explored administration of the nanoparticles via a non-occluded carotid artery as a way to increase the passive exposure of tumor vasculature to nanoparticles for subsequent magnetic entrapment. However, aggregation of nanoparticles in the afferent vasculature interfered with tumor targeting. The magnetic setup employed in our experiments was found to generate a relatively uniform magnetic flux density over a broad range, exposing the region of the afferent vasculature to high magnetic force. To overcome this problem, the magnetic setup was modified with a 9-mm diameter cylindrical NdFeB magnet to exhibit steeper magnetic field topography. Six-fold reduction of the magnetic force at the injection site, achieved with this modification, alleviated the aggregation problem under the conditions of intact carotid blood flow. Using this setup, carotid administration was found to present 1.8-fold increase in nanoparticle accumulation in glioma compared to the intravenous route at 350mT. This increase was found to be in reasonable agreement with the theoretically estimated 1.9-fold advantage of carotid administration, R(d). The developed approach is expected to present an even greater advantage when applied to drug-loaded nanoparticles exhibiting higher values of R(d).

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL)

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

The magnetophoretic mobility and superparamagnetism of core-shell iron oxide nanoparticles with dual targeting and imaging functionality.

With the goal to achieve highly efficacious MRI-monitored magnetic targeting, a novel drug carrier with dual nature of superior magnetophoretic mobility and superparamagnetism was synthesized. This carrier was specially designed in a core-shell structure. The core was achieved by utilizing a strategy of self-assembly of oppositely charged ultrafine superparamagnetic iron oxide nanoparticles previously prepared. The final particles were formed by coating such cores with carboxymethyldextran (CMD) polymer. By exclusion of non-magnetic materials from the interior part of the particles, this structure maximized the amount of magnetic material and thus yielded a superior magnetophoretic mobility. Such a strategy avoids the challenge of superparamagnetism loss, which would be caused by cores exceeding a critical domain size. Coating the self-assembled core enables the magnetic carrier to be stable upon usage and storage and to be readily linked with drug molecules for therapeutic applications. In vitro characterization showed that these nanoparticles displayed a 3- to 4-fold enhancement in magnetophoretic mobility, and a markedly improved stability when stored in 50% serum as a comparison of conventional iron oxide-based magnetic nanoparticles. Preliminary in vivo studies revealed that the nanoparticles also function well as a contrast enhancer for MR imaging of brain glioma. This technology could lead to the development of a new paradigm of magnetic carriers that meet with the needs of various clinical applications.

Comparison of Electron Spin Resonance Spectroscopy and Inductively-Coupled Plasma Optical Emission Spectroscopy for Biodistribution Analysis of Iron-Oxide Nanoparticles

Magnetic nanoparticles (MNP) have been widely studied for use in targeted drug delivery. Analysis of MNP biodistribution is essential to evaluating the success of targeting strategies and the potential for off-target toxicity. This work compared the applicability of inductively coupled plasma optical emission spectroscopy (ICP-OES) and electron spin resonance (ESR) spectroscopy in assessing MNP biodistribution. Biodistribution was evaluated in 9L-glioma bearing rats administered with MNP (12-25 mg Fe/kg) under magnetic targeting. Ex vivo analysis of MNP in animal tissues was performed with both ICP-OES and ESR. A cryogenic method was developed to overcome the technical hurdle of loading tissue samples into ESR tubes. Comparison of results from the ICP-OES and ESR measurements revealed two distinct relationships for organs accumulating high or low levels of MNP. In organs with high MNP accumulation such as the liver and spleen, data were strongly correlated (r = 0.97, 0.94 for the liver and spleen, respectively), thus validating the equivalency of the two methods in this high concentration range (>1000 nmol Fe/g tissue). The two sets of measurements, however, differed significantly in organs with lower levels of MNP accumulation such as the brain, kidney, and the tumor. Whereas ESR resolved MNP to 10-55 nmol Fe/g tissue, ICP-OES failed to detect MNP because of masking by endogenous iron. These findings suggest that ESR coupled to cryogenic sample handling is more robust than ICP-OES, attaining better sensitivity in analyses. Such advantages render ESR the method of choice for accurate profiling of MNP biodistribution across tissues with high variability in nanoparticle accumulation.

Substantiating In Vivo Magnetic Brain Tumor Targeting of Cationic Iron Oxide Nanocarriers via Adsorptive Surface Masking

Cationic magnetic nanoparticles are attractive as potential vehicles for tumor drug delivery due to their favorable interactions with both the tumor milieu and the therapeutic cargo. However, systemic delivery of these nanoparticles to the tumor site is compromised by their rapid plasma clearance. We developed a simple method for in vivo protection of cationic nanocarriers, using non-covalent surface masking with a conjugate of low molecular weight heparin and polyethylene glycol. Surface masking resulted in a 11-fold increase in plasma AUC and a 2-fold increase in the magnetic capture of systemically injected nanoparticles in orthotopic rodent brain tumors. Overall, the described methodology could expand the prospective applications for cationic magnetic nanoparticles in magnetically mediated gene/drug delivery.

Magnetic Nanoparticles for Tumor Imaging and Therapy: A So-Called Theranostic System

ABSTRACTIn this review, we discussed the establishment of a so-called “theranostic” system by instituting the basic principles including the use of: [1] magnetic iron oxide nanoparticles (MION)-based drug carrier; [2] intra-arterial (I.A.) magnetic targeting; [3] macromolecular drugs with unmatched therapeutic potency and a repetitive reaction mechanism; [4] cell-penetrating peptide-mediated cellular drug uptake; and [5] heparin/protamine-regulated prodrug protection and tumor-specific drug re-activation into one single drug delivery system to overcome all possible obstacles, thereby achieving a potentially non-invasive, magnetic resonance imaging-guided, clinically enabled yet minimally toxic brain tumor drug therapy. By applying a topography-optimized I.A. magnetic targeting to dodge rapid organ clearance of the carrier during its first passage into the circulation, tumor capture of MION was enriched by >350 folds over that by conventional passive enhanced permeability and retention targeting. By adopting the prodrug strategy, we observed by far the first experimental success in a rat model of delivering micro-gram quantity of the large β-galactosidase model protein selectively into a brain tumor but not to the ipsi- or contra-lateral normal brain regions. With the therapeutic regimens of most toxin/siRNA drugs to fully (>99.9%) eradicate a tumor being in the nano-molar range, the prospects of reaching this threshold become practically accomplishable.

Synthetic Skin-Permeable Proteins Enabling Needleless Immunization†

Protein drugs, due to their large size and hydrophilic nature, are normally precluded from effective delivery such as cell entry or tissue diffusion. Among the transport barriers, the skin poses as a formidable challenge to proteins due to the impermeable stratum corneum. The existing techniques for percutaneous protein delivery must rely on sophisticated delivery systems, such as the use of complicated nanocarriers or mechanical devices, to overcome the skin barrier for noninvasive delivery. A challenge in manufacturing of such systems is their complicated processes and potential negative impact on protein drug stability. Moreover, the high manufacturing cost of these advanced systems often offsets their remarkable advantages. To circumvent these problems that confront the current methods, we hypothesized the concept of “skin-permeable proteins” which would possess skin penetrating ability, and thereby eliminate a need for a transport vehicle. However, naturally occurring proteins with skin penetrating ability rarely exist. Herein, we present a novel strategy for chemically constructing artificial skin-permeable proteins, featured by a simple conjugation of a protein to a cell-penetrating peptide (CPP), which would display a penetration effect on the stratum corneum barrier, and transport the attached proteins into the skin. Furthermore, the feasibility of application in transcutaneous immunization is demonstrated. CPPs are known for their versatility in carrying macro- or supra-molecules through the cell membrane barriers that challenge the conventional drug delivery approaches.[1] The CPPs are capable of transporting their cargos, often linked by a covalent bond, into almost all cell types.[2] Among such CPPs, the low molecular weight protamine (LMWP) peptide (VSRRRRRRGGRRRRR), developed in our laboratory by enzymatic digestion of protamine (an FDA approved drug), offers distinct advantages. First, LMWP is as potent as the virus-derived TAT peptide, the most-studied CPP to date, in mediating cellular translocation of the attached cargos.[3] Secondly, unlike other CPPs, the toxicity profile of LMWP has already been thoroughly established. LMWP was shown to be non-immunogenic,[4] and its use in dogs did not elicit acute toxic responses.[5] Lastly, while other CPPs must be chemically synthesized, LMWP can be produced in mass quantities direct from native protamine with limited processing time and cost.[6] In this investigation, the artificial skin-permeable protein was synthesized by conjugating LMWP to ovalbumin (OVA), a representative antigenic protein, via a cleavable disulfide bond (Scheme 1). The LMWP-OVA conjugates were purified by heparin affinity chromatography, and the final product, generally possessing a 1:1 molar ratio of LMWP:OVA, was verified by MALDI-TOF-MS. Scheme 1 Chemical conjugation of LMWP to OVA As noted, skin keratinocytes are a physical barrier that provides the front line of defense against infection and also poses a challenge to protein delivery. On the other hand, keratinocytes execute a “part-time” antigen-presenting function by secreting immune mediators and transferring antigens to local antigen-presenting cells.[7] LMWP was shown to exhibit an ability to translocate the linked cargos of varying sizes into keratinocytes (Figure 1), demonstrating the potential for percutaneous protein delivery. Figure 1 Uptake by human keratinocyte cells of a) rhodamine B; b) OVA; and c) BSA; compared with those of d) LMWP-rhodamine B; e) LMWP-OVA; and f) LMWP-BSA conjugates. Protein cargos were labeled with FITC. (Scale bar = 100 μm) The plausibility of percutaneous delivery in vivo was examined by topical application of LMWP-linked lysozyme, OVA, or bovine serum albumin (BSA), to represent a broad range of protein sizes. All the LMWP-linked proteins successfully penetrated the stratum corneum and accumulated primarily in the epidermis (Figure 2), whereas the control proteins without LMWP linkage remained on the surface of skin. Figure 2 In vivo transcutaneous delivery mediated by LMWP. a), b), and c) represented unmodified free lysozyme, OVA, and BSA, whereas d), e), and f) represented LMWP-linked lysozyme, OVA, and BSA, respectively. Arrows represented the direction of skin penetration. ... The skin penetration mechanism of CPPs is still under debate. However, the interaction between CPP and lipid bilayer is believed to play a major role in the cell penetration process.[8] The skin permeability is governed by the physical state and structural organization of the extracellular lipids.[9] Hence, the skin penetration function of LMWP could account for its interaction with the skin extracellular lipid matrices. Such interaction would lead to disruption of the ordered lipid orientation, thereby creating channels for transducing protein cargos through the stratum corneum. As a typical example of protein percutaneous delivery, the immunological milieu of the skin is an ideal site for noninvasive vaccine delivery. The epidermis is rich in mature Langerhans cells (LCs), which represent a network of immune cells that underlie 25% of the total surface area in human skin,[10] and thus the epidermis is the target skin layer for transcutaneous immunization (TI). TI can be achieved by topically applying antigens, which, with the aid of a transdermal delivery system, penetrate into skin and subsequently elicit the desired immunity. The network of LCs acts as an immunological line of defense and initiates immune responses by conveying the captured antigens to other cells of the immune system, e.g. lymphocytes, melanocytes and Mercel cells.[11] Therefore, the unique epidermal accumulation of the LMWP-linked proteins offers an ideal situation to alert such antigen-presenting cells. The constructed skin-permeable antigen of LMWP-OVA was tested for the feasibility of TI on Balb/c mice. Humoral IgG is the primary protection induced by preventive vaccines to neutralize and eliminate of pathogens. Figure 3a revealed that a significant elevation of anti-OVA IgG concentration in the blood was observed following topical application of LMWP-OVA with cholera toxin as adjuvant. The IgG levels in TI groups treated with the high- (TI-H) and medium-dose (TI-M) of antigen displayed no statistical differences (p > 0.05) from those in animals given OVA through the standard intramuscular immunization method (IM group). The control group, receiving topical native OVA, exhibited markedly lower levels of IgG, due to poor percutaneous absorption of unmodified OVA. These findings indicated that the epidermis-accumulated LMWP-OVA was captured by LCs that subsequently migrated to lymphoid tissues and presented the antigens, effectively eliciting robust humoral immune responses. Furthermore, disulfide linkage could be cleaved by the elevated level of glutathione and reductase activity in the cytosol,[12] allowing release of OVA from LMWP, thus retaining a full intrinsic immunogenicity. As evidence, LMWP-OVA in TI method triggered OVA-specific IgG responses comparable to the IM injection of OVA. Since the conjugation of LMWP to OVA might affect its intrinsic antigenic determinants, a cleavable linkage could ease such concern. Figure 3 Transcutaneous immunization study. Mice were topically immunized with high- (500 μg; TI-H), medium- (250 μg; TI-M), and low-dose (100 μg; TI-L) of LMWP-OVA. a) High levels of anti-OVA IgG were observed in all TI groups, with no ... TI shows advantages over conventional injection vaccination by offering the opportunity to elicit specific immune responses, such as targeted immunity to the female reproductive tract[13] and cytotoxic T lymphocytes (CTL) effect.[14] Secretory IgA (sIgA) is the predominant humoral defense mechanism at mucosal surface, and it therefore protects the host from initial infections. As shown in Figure 3b, the anti-OVA sIgA levels measured in vaginal secretions were significantly higher in TI-H and TI-M groups than those in the IM group, confirming the promise of TI in achieving local protective immunity against female genital infection. Furthermore, interferon-γ(IFN-γ), the representative cytokine known to enhance the CD8+ CTL-mediated cytotoxicity against infected cells, was also present at a level significantly higher in the TI groups than in the IM group (Figure 3c). Notably, local immune response in skin could also benefit from production of high levels of IFN-γ, due to its effect on promoting CTL recognition of antigen molecules in keratinocytes[15] and subsequently their expedited lysis.[16] In addition, a primer-booster vaccination conducted by combining the IM injection of OVA with transcutaneous boosters of LMWP-OVA showed the immunity induction comparable to the multi-shot IM standard method (Figure S 1). The self-administrable boosters would eliminate follow-up visits to clinics for a multi-dose protocol. Hence this immunization strategy could improve not only patient compliance but also vaccination coverage in underserved areas with limited medical settings. In conclusion, this methodology for constructing artificial skin-permeable antigens may offer simple and needle-free vaccination modalities without the need for sophisticated drug carriers or expensive medical devices. Such a method could be beneficial especially to developing countries that struggle to fulfill effective vaccination coverage.

Magnetically-enabled and MR-monitored selective brain tumor protein delivery in rats via magnetic nanocarriers

The delivery of bioactive proteins to tumors is associated with many difficulties that have impeded clinical translation of these promising therapeutics. Herein we present an approach, including (1) use of magnetically-responsive and MRI-visible nanoparticles as drug carriers, (2) topography-optimized intra-arterial magnetic targeting, (3) MRI-guided subject alignment within the magnetic field, and (4) surface modification of the protein drug with membrane-permeable polyethyleneimine (PEI), to prevail over the obstacles in protein delivery. Applying these methodologies, we demonstrated the delivery of a significant quantity of β-galactosidase selectively into brain tumors of glioma-bearing rats, while limiting the exposure of normal brain regions. Clinical viability of the technologies utilized, and the ability to deliver proteins at high nanomolar-range tumor concentrations, sufficient to completely eradicate a tumor lesion with existing picomolar-potency protein toxins, renders the prospect of enabling protein-based cancer therapy extremely promising.