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Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand., Competing Interests: All authors are past or current employees of AstraZeneca., (© 2024 AstraZeneca. Published by National Institute for Materials Science in partnership with Taylor & Francis Group.)

Despite advances by recently approved antibody-drug conjugates in treating advanced gastric cancer patients, substantial limitations remain. Here, several key obstacles are overcome by developing a first-in-class ultrasmall (sub-8-nanometer (nm)) anti-human epidermal growth factor receptor 2 (HER2)-targeting drug-immune conjugate nanoparticle therapy. This multivalent fluorescent core-shell silica nanoparticle bears multiple anti-HER2 single-chain variable fragments (scFv), topoisomerase inhibitors, and deferoxamine moieties. Most surprisingly, drawing upon its favorable physicochemical, pharmacokinetic, clearance, and target-specific dual-modality imaging properties in a "hit and run" approach, this conjugate eradicated HER2-expressing gastric tumors without any evidence of tumor regrowth, while exhibiting a wide therapeutic index. Therapeutic response mechanisms are accompanied by the activation of functional markers, as well as pathway-specific inhibition. Results highlight the potential clinical utility of this molecularly engineered particle drug-immune conjugate and underscore the versatility of the base platform as a carrier for conjugating an array of other immune products and payloads., Competing Interests: Conflict of Interest M.S.B., U.W., F.C., K.M., and M.Z.T. hold interest in, and U.W. a board seat of, Elucida Oncology, Inc., which has licensed IP from Cornell and MSK on C′ dots and their application in oncology. The remaining authors declare no conflict of interest.

Optimizing the Diels-Alder (DA) reaction for aqueous coupling has resulted in practical methods to link molecules such as drugs and diagnostic agents to proteins. Both normal electron demand (NED) and inverse electron demand (IED) DA coupling schemes have been employed, but neither mechanism entails a common multipurpose reactive group. This report focuses on expanding the bioconjugation toolbox for cyclopentadiene through the identification of reactive groups that couple through NED or IED mechanisms in aqueous solution. Dienophiles and tetrazine derivatives were screened for reactivity and selectivity toward antibodies bearing cyclopentadiene amino acids to yield bioconjugates. Twelve NED dienophiles and four tetrazine-based IED substrates were identified as capable of practical biocoupling. Furthermore, tetrazine ligation to cyclopentadiene occurred at a rate of 3.3 ± 0.5 M -1 s -1 and was capable of bioorthogonal transformations, as evidenced by the selective protein labeling in serum. Finally, an antibody-drug conjugate (ADC)-bearing monomethyl auristatin E was prepared via tetrazine conjugation to cyclopentadiene. The resulting ADC was stable and demonstrated potent activity in vitro . These findings expand the utility of cyclopentadiene as a tool to couple entities to proteins via dual DA addition mechanisms.

Assessment of drug activation and subsequent interaction with targets in living tissues could guide nanomedicine design, but technologies enabling insight into how a drug reaches and binds its target are limited. We show that a Hoechst-based reporter system can monitor drug release and engagement from a nanoparticle delivery system in vitro and in vivo , elucidating differences in target-bound drug distribution related to drug-linker and nanoparticle properties. Drug engagement is defined as chemical detachment of drug or reporter from a nanoparticle and subsequent binding to a subcellular target, which in the case of Hoechst results in a fluorescence signal. Hoechst-based nanoreporters for drug activation contain prodrug elements such as dipeptide linkers, conjugation handles, and nanoparticle modifications such as targeting ligands to determine how nanomedicine design affects distribution of drug engaged with a subcellular target, which is tracked via cellular nuclear fluorescence in situ . Furthermore, the nanoplatform is amenable toward common maleimide-based linkers found in many prodrug-based delivery systems including polymer-, peptide-, and antibody-drug conjugates. Findings from the Hoechst reporter system were applied to develop highly potent, targeted, anticancer micelle nanoparticles delivering a monomethyl auristatin E (MMAE) prodrug comprising the same linkers employed in Hoechst studies. MMAE nanomedicine with the optimal drug-linker resulted in effective tumor growth inhibition in mice without associated acute toxicity, whereas the nonoptimal linker that showed broader drug activation in Hoechst reporter studies resulted in severe toxicity. Our results demonstrate the potential to synergize direct visualization of drug engagement with nanomedicine drug-linker design to optimize safety and efficacy.

Successful gene therapies rely on methods that safely introduce DNA into target cells and enable subsequent expression of proteins. To that end, peptides are an attractive materials platform for DNA delivery, facilitating condensation into nanoparticles, delivery into cells, and subcellular release to enable protein expression. Peptides are programmable materials that can be designed to address biocompatibility, stability, and subcellular barriers that limit efficiency of non-viral gene delivery systems. This review focuses on fundamental structure-function relationships regarding peptide design and their impact on nanoparticle physical properties, biologic activity, and biocompatibility. Recent peptide technologies utilize multi-dimensional structures, non-natural chemistries, and combinations of peptides with lipids to achieve desired properties and efficient transfection. Advances in DNA cargo design are also presented to highlight further opportunities for peptide-based gene delivery. Modern DNA designs enable prolonged expression compared to traditional plasmids, providing an additional component that can be synergized with peptide carriers for improved transfection. Peptide transfection systems are poised to become a flexible and efficient platform incorporating new chemistries, functionalities, and improved DNA cargos to usher in a new era of gene therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following competing financial interests: MU and RJC are current employees of AstraZeneca., (Copyright © 2020. Published by Elsevier Ltd.)

Synthetic gene delivery systems employ multiple functions to enable safe and effective transport of DNA to target cells. Here, we describe metabolite-based poly(l-lysine) (PLL) modifiers that improve transfection by imparting both pH buffering and nanoparticle stabilization functions within a single molecular unit. PLL modifiers were based on morpholine (M), morpholine and niacin (MN), or thiomorpholine (TM). PLL modification with (MN) or (TM) imparted buffering function over the pH range of 5-7 both in solution and live cells and enhanced the stability of PLL DNA nanoparticles, which exhibited higher resistance to polyanion exchange and prolonged blood circulation. These properties translated into increased transfection efficiency in vitro coupled with reduced toxicity compared to unmodified PLL and PLL(M). Furthermore, PEG-PLL(MN) DNA nanoparticles transfected muscle tissue in vivo for >45 days following intramuscular injection. These polymer modifiers demonstrate the successful design of multifunctional units that improve transfection of synthetic gene delivery systems while maintaining biocompatibility.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M -1 s -1 ), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

Here, we describe a diene-containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron-demand Diels-Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self-reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug-linker AZ1508 at a rate of ≈79 m -1  s -1 to produce functional antibody-drug conjugates (ADCs) in a one-step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity-based stapling through a DA dimerization reaction., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors and hematological malignancies, improvements in ADC design are still desirable due to the narrow therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of many different types of biologics. PEGylation improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates. Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the mechanism of half-life extension is an important consideration for designing cytotoxic antitumor agents.

The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors., Competing Interests: CONFLICTS OF INTEREST Jiping Zha is currently an employee of NGM Pharmaceuticals, Joanne Ayriss is currently an employee of Pfizer, Partha Chowdhury is currently an employee of Sanofi Genzyme, Binyam Bezabeh is currently an employee of Salubris Biotherapeutics. All additional authors are employees of MedImmune, LLC.

Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N -phenyl maleimide functionality for stable thiol conjugation via thiosuccinimide hydrolysis. Two PBD drug-linker formats (enzyme-cleavable and non-cleavable) were evaluated following site-specific conjugation to an engineered cysteine incorporated at position T289, which is known to be unstable for N -alkyl maleimide conjugates. N -phenyl maleimide PBDs conjugated to antibodies with similar efficiencies as N -alkyl maleimide PBDs and enhanced thiosuccinimide hydrolysis for N -phenyl maleimide PBDs was confirmed by mass spectrometry, capillary isoelectric focusing, and a SYPRO Orange dye binding assay. All of the PBD ADCs were highly potent in vitro regardless of maleimide- or linker-type, exhibiting low pM EC 50 values. Thiol conjugation to N -phenyl maleimide PBD minimized the retro-Michael reaction in both rat and mouse serum. However, cleavage of the valine-alanine dipeptide in mouse serum for ADCs containing cleavable drug-linker led to drug loss regardless of maleimide type, which impacted ADC potency in tumor growth inhibition studies that were conducted in mouse models. Therapeutic improvement in mouse tumor models was realized for ADCs prepared with non-cleavable PBD drug-linkers that were conjugated through N -phenyl maleimide, where a stronger tumor growth inhibition (TGI) response was achieved when compared to the analogous N -alkyl maleimide drug-linker ADC. Altogether, our findings highlight the stability and efficacy benefits of N -phenyl maleimide functionality for ADCs that are produced with thiol-maleimide conjugation chemistry., Competing Interests: The authors declare the following competing financial interest: all authors are current or previous employees of the AstraZeneca Group and work to develop ADCs for clinical applications.

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) E6 and E7 oncogenes are essential for the immortalization and maintenance of HPV-associated cancer and are ubiquitously expressed in cervical cancer lesions. Small interfering RNA (siRNA) coding for E6 and E7 oncogenes is a promising approach for precise treatment of cervical cancer, yet a delivery system is required for systemic delivery to solid tumors. Here, an actively targeted polyion complex (PIC) micelle was applied to deliver siRNAs coding for HPV E6/E7 to HPV cervical cancer cell tumors in immune-incompetent tumor-bearing mice. A cell viability assay revealed that both HPV type 16 and 18 E6/E7 siRNAs (si16E6/E7 and si18E6/E7, respectively) interfered with proliferation of cervical cancer cell lines in an HPV type-specific manner. A fluorescence imaging biodistribution analysis further revealed that fluorescence dye-labeled siRNA-loaded PIC micelles efficiently accumulated within the tumor mass after systemic administration. Ultimately, intravenous injection of si16E6/E7 and si18E6/E7-loaded PIC micelles was found to significantly suppress the growth of subcutaneous SiHa and HeLa tumors, respectively. The specific activity of siRNA treatment was confirmed by the observation that p53 protein expression was restored in the tumors excised from the mice treated with si16E6/E7- and si18E6/E7-loaded PIC micelles for SiHa and HeLa tumors, respectively. Therefore, the actively targeted PIC micelle incorporating HPV E6/E7-coding siRNAs demonstrated its therapeutic potential against HPV-associated cancer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Enhanced permeability in tumours is thought to result from malformed vascular walls with leaky cell-to-cell junctions. This assertion is backed by studies using electron microscopy and polymer casts that show incomplete pericyte coverage of tumour vessels and the presence of intercellular gaps. However, this gives the impression that tumour permeability is static amid a chaotic tumour environment. Using intravital confocal laser scanning microscopy we show that the permeability of tumour blood vessels includes a dynamic phenomenon characterized by vascular bursts followed by brief vigorous outward flow of fluid (named 'eruptions') into the tumour interstitial space. We propose that 'dynamic vents' form transient openings and closings at these leaky blood vessels. These stochastic eruptions may explain the enhanced extravasation of nanoparticles from the tumour blood vessels, and offer insights into the underlying distribution patterns of an administered drug.

Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

Ballistic delivery capability is essential to delivering vaccines and other therapeutics effectively to both livestock and wildlife in many global scenarios. Here, lyophilized poly(ethylene glycol) (PEG)-glycolide dimethacrylate crosslinked but degradable hydrogels were assessed as payload vehicles to protect and deliver a viable bacterial vaccine, Brucella abortus strain RB51 (RB51), ballistically using commercial thermoplastic cellulosic degradable biobullets. Degradable PEG hydrogel rods loaded with ∼10(10) live RB51 bacteria (CFUs) were fabricated using three different polymerization methods, cut into fixed-sized payload segments, and lyophilized. Resulting dense, glassy RB51 vaccine-loaded monoliths were inserted into thermoplastic biobullet 100-μL payload chambers. Viability studies of lyophilized formulations assessed as a function of time and storage temperature supported the abilities of several conditions to produce acceptable vaccine shelf-lives. Fired from specifically designed air rifles, gel-loaded biobullets exhibit down-range ballistic properties (i.e., kinetic energy, trajectory, accuracy) similar to unloaded biobullets. Delivered to bovine tissue, these hydrogels rehydrate rapidly by swelling in tissue fluids, with complete hydration observed after 5h in serum. Live RB51 vaccine exhibited excellent viability following carrier polymerization, lyophilization, and storage, at levels sufficient for vaccine dosing to wild range bison, the intended target. These data validate lyophilized degradable PEG hydrogel rods as useful drug carriers for remote delivery of both live vaccines and other therapeutics to livestock, wildlife, or other free-range targets using ballistic technologies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Maleimides are often used to covalently attach drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug. Here, we report that N-aryl maleimides form stable antibody conjugates under very mild conditions while also maintaining high conjugation efficiency. Thiol-maleimide coupling and ADC stabilization via thiosuccinimide hydrolysis were accelerated by addition of N-phenyl or N-fluorophenyl groups to the ring-head nitrogen. Cysteine-linked ADCs prepared with N-aryl maleimides exhibited less than 20% deconjugation in both thiol-containing buffer and serum when incubated at 37 °C over a period of 7 days, whereas the analogous ADCs prepared with N-alkyl maleimides showed 35-67% deconjugation under the same conditions. ADCs prepared with the anticancer drug N-phenyl maleimide monomethyl-auristatin-E (MMAE) maintained high cytotoxicity following long-term exposure to serum whereas the N-alkyl maleimide MMAE ADC lost potency over time. These data demonstrate that N-aryl maleimides are a convenient and flexible platform to improve the stability of ADCs through manipulation of functional groups attached to the maleimide ring-head nitrogen., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hydrophobized block copolymers have widely been developed for construction of polymeric micelles for stable delivery of nucleic acids as well as anticancer drugs. Herein, we elaborated an A-B-C type of triblock copolymer featuring shell-forming A-segment, nucleic acid-loading B-segment, and stable core-forming C-segment, directed toward construction of a three-layered polymeric micelle as a small interfering RNA (siRNA) vehicle. The triblock copolymer was prepared with nonionic and hydrophilic poly(ethylene glycol) (PEG), cationic poly(l-lysine) (PLys), and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] bearing a hydrophobic dimethoxy nitrobenzyl ester (DN) moiety in the side chain [PEG-PLys-PAsp(DET-DN)]. The resulting triblock copolymers spontaneously formed sub-100 nm-sized polymeric micelles with a hydrophobic PAsp(DET-DN) core as well as PEG shell in an aqueous solution. This micelle was able to incorporate siRNA into the intermediate PLys layer, associated with slightly reduced size and a narrow size distribution. The triblock copolymer micelles (TCMs) stably encapsulated siRNA in serum-containing medium, whereas randomly hydrophobized triblock copolymer [PEG-PLys(DN)-PAsp(DET-DN)] control micelles (RCMs) gradually released siRNA with time and non-PEGylated diblock copolymer [PLys-PAsp(DET-DN)] control micelles (DCMs) immediately formed large aggregates. The TCMs thus induced appreciably stronger sequence-specific gene silencing in cultured cancer cells, compared to those control micelles. The siRNA delivery with TCMs was further examined in terms of cellular uptake and intracellular trafficking. The flow cytometric analysis revealed that the cellular uptake of TCMs was more efficient than that of RCMs, but less efficient than that of DCMs. The intracellular trafficking study using confocal laser scanning microscopy combined with fluorescence resonance energy transfer (FRET) revealed that the TCMs could readily release the siRNA payload within cells, which was in contrast to the DCMs exhibiting much slower release profile. This result indicates that PEG shell contributed to the smooth release of siRNA from TCMs within the cells, presumably due to avoiding irreversible aggregate formation. The obtained results demonstrated that the design of separately functionalized polymer segments expanded the performance of polymeric micelles for successful siRNA delivery., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we describe a multifunctional, nontoxic delivery vehicle with dual-environment sensitivity to deliver plasmid DNA (pDNA) into the cytoplasm of cells. This delivery vehicle was designed to be destabilized by reduction of disulfide cross-links in the intracellular environment and also to contain pH-sensitive membrane-destabilizing activity in acidic late endosomal/lysosomal compartments to allow escape of pDNA into the cell cytoplasm. Polyion complex formation was used to form ternary polyplexes using ionic polymers containing specific chemistries to achieve functional demands. First, template binary polyplexes were formed by association of cationic poly(l-lysine) containing thiol groups (PLys(PDP)) with pDNA and were subsequently cross-linked by disulfide formation for increased stability. Then, binary cross-linked polyplexes were coated with a pH-sensitive membrane-active polyanion, poly(ethylene glycol)-b-poly(aspartamide(DET-Aco)) (PEG-PAsp(DET-Aco)), to produce ternary cross-linked polyplexes. PEG-PAsp(DET-Aco) comprises two repeating units of aminoethylene in PAsp side chains and primary amines modified with anionic cis-aconitic groups. PEG-PAsp(DET-Aco) degrades at acidic pH to generate the parent PEG-PAsp(DET) polymer, which is active toward late endosomal/lysosomal membranes and thus can assist in the endosomal escape of pDNA following endocytosis. Binary/ternary cross-linked polyplexes remained stable toward counter polyanion exchange with dextran sulfate, but released pDNA following disulfide reduction. Ternary cross-linked polyplexes formed by addition of PEG-PAsp(DET-Aco) resulted in enhanced gene transfection efficiency in cultured cells (Huh-7 and HUVEC) without associated cytotoxicity. The enhanced gene transfection was found to be correlated with improved endosomal escape by observation of intracellular trafficking using confocal laser scanning microscopy. This multifunctional ternary cross-linked polyplex demonstrates the successful design of a gene delivery vehicle utilizing intracellular stimuli, and is a promising platform for further development toward practical use.

Multifunctional delivery systems of small interfering RNA (siRNA) are needed to overcome the intrinsic biological barriers toward efficient gene silencing in the cell cytoplasm. In this report, a smart multilayered assembly (SMA) was fabricated by a layer-by-layer method with polyionic materials. The SMA was designed to feature a siRNA-loaded core, a transiently core-stabilizing silica interlayer, an endosome-disrupting polycation interlayer, and a biocompatible poly(ethylene glycol) (PEG) shell with reductive environment-responsive detachability. The SMA was confirmed to be approximately 160 nm in size with narrow distribution and spherical morphology by DLS and TEM analyses. The PEG detachability of the SMA based on disulfide cleavage was also confirmed by the increase in both ζ-potential and size due to the exposure of the polycation interlayer and the compromised colloidal stability. The silica interlayer rendered the SMA highly tolerant to dissociation induced by anionic lipids, while after 24 h dialysis siRNA release from the SMA was clearly observed, presumably due to gradual dissolution of the silica interlayer based on the equilibrium shift to silicate ions. The entrapment ratio of siRNA delivered by the SMA within the endosome was significantly lower than that by nondisulfide control (NDC) without PEG detachability, suggesting the improved endosomal escape of SMA with the exposed, endosome-disrupting interlayer after PEG detachment. SMAs induced significantly higher gene silencing efficiency in various cultured cells, compared to NDC, without associated cytotoxicity. The systemic administration of SMAs for subcutaneous tumor-bearing mice achieved significant endogenous gene silencing in tumor tissue without hematological toxicity.

Development of an efficient in vivo delivery vehicle of small interfering RNA (siRNA) is the key challenge for successful siRNA-based therapies. In this study, toward systemic delivery of siRNA to solid tumors, a smart polymer/calcium phosphate (CaP)/siRNA hybrid nanoparticle was prepared to feature biocompatibility, reversible stability and endosomal escape functionality using a pH sensitive block copolymer of poly(ethylene glycol) and charge-conversional polymer (PEG-CCP), of which anionic functional groups could be converted to cationic groups in an endosomal acidic condition for facilitated endosomal escape. Nanoparticles were confirmed to be approximately 100nm in size, narrowly dispersed and spherical. Also, the nanoparticle was highly tolerable in medium containing serum, while releasing the entrapped siRNA in a cytoplasm-mimicking ionic condition, presumably based on the equilibrium between CaP complexes and calcium ions. Further, the nanoparticle showed high gene silencing efficiency in cultured pancreatic cancer cells (BxPC3) without associated cytotoxicity. Ultimately, systemic administration of the nanoparticles carrying vascular endothelium growth factor (VEGF) siRNA led to the significant reduction in the subcutaneous BxPC3 tumor growth, well consistent with the enhanced accumulation of siRNA and the significant VEGF gene silencing (~68%) in the tumor. Thus, the hybrid nanoparticle was demonstrated to be a promising formulation toward siRNA-based cancer therapies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Small interfering ribonucleic acid (siRNA) cancer therapies administered by intravenous injection require a delivery system for transport from the bloodstream into the cytoplasm of diseased cells to perform the function of gene silencing. Here we describe nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures on the order of 45 nm in size formed by spontaneous self-assembly of block copolymers with siRNA. Block copolymers used for micelle formation were designed and synthesized to contain three main features: a siRNA binding segment containing thiols, a hydrophilic nonbinding segment, and a cell-surface binding peptide. Specifically, poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) comprising lysine amines modified with 2-iminothiolane (2IT) and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus was used. Modification of PEG-b-PLL with 2IT led to improved control of micelle formation and also increased stability in the blood compartment, while installation of the cRGD peptide improved biological activity. Incorporation of siRNA into stable micelle structures containing the cRGD peptide resulted in increased gene silencing ability, improved cell uptake, and broader subcellular distribution in vitro and also improved accumulation in both the tumor mass and tumor-associated blood vessels following intravenous injection into mice. Furthermore, stable and targeted micelles inhibited the growth of subcutaneous HeLa tumor models and demonstrated gene silencing in the tumor mass following treatment with antiangiogenic siRNAs. This new micellar nanomedicine could potentially expand the utility of siRNA-based therapies for cancer treatments that require intravenous injection.

Selective packaging of plasmid DNA (pDNA) into folded rod or collapsed sphere structures in polyplex micelles was demonstrated by modulating the PLys segment length of poly(ethylene glycol)-block- poly(L-lysine) (PEG-PLys) block catiomers used for micelle formation. The two basic packaging structures correlated well to the integrity of double-stranded DNA contained within the micelles. Rod structures formed by the quantized folding mechanism, which results in dissociation of double-stranded DNA only at each fold. Collapsed sphere structures formed by substantial random disruption of the double-stranded DNA structure. Analysis of gene expression in a cell-free transcription/translation system, cultured cells and also skeletal muscle of mice showed that micelles containing pDNA packaged by quantized folding exhibited higher gene expression than naked pDNA and micelles containing collapsed pDNA. These results indicate that controlled packaging of pDNA into an appropriate structure is critical for achieving effective gene expression. Improved gene transfection and expression resulting from the quantized folding of pDNA within polyplex micelles is promising for application in therapeutic gene delivery systems., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The concept of drug delivery systems (DDS) promises the treatments for the refractory diseases such as cancer. Many kinds of multifunctional DDS have been developed, demonstrating enhanced drug accumulation and therapeutic efficiency. However, it is quite difficult to evaluate that the DDS really perform as expected under in vivo conditions. We have recently developed the intravital real-time confocal laser scanning microscopy (IVRTCLSM) characterized by rapid scanning and simultaneous multicolor detection, to visualize the behavior of DDS in living mice. IVRTCLSM revealed the dynamic states of DDS, which could not be observed by conventional in vitro/ex vivo methods, in the bloodstream, tumors, and other organs. IVRTCLSM will provide new facets in the DDS study, and will facilitate the development of DDS.

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.

Case History: In March 2006, an outbreak of photosensitivity affecting 6-8-month-old Friesian heifer calves on a farm in the Rangitikei district of New Zealand was investigated. The calves were grazing wheat stubble paddocks that also had a variety of weeds, especially Phytolacca octandra (inkweed). They also had access to pond water that contained potentially toxic concentrations of the cyanobacteria (blue-green algae) Microcystis and Planktothrix spp., Clinical Findings: Initially, affected animals showed acute irritation, agitation, reluctance to walk, recumbency in some animals, hyperaemia of unpigmented skin and jaundice. Serum chemistry revealed elevated liver enzyme activities and azotaemia. Later in the outbreak, exudative dermatitis with formation of crusts on unpigmented skin, dehydration and inappetence were notable signs, as well as occasional diarrhoea. PATHOLOGICAL AND TOXICOLOGICAL FINDINGS: Post-mortem examinations following euthanasia of four severely affected calves and a fifth animal that died naturally revealed livers that were grossly orange brown and mildly enlarged, and kidneys that were enlarged and pale brown. Microscopic lesions in the liver were mild; however, small birefringent crystals in the bile ducts were noted in the first two cases. In contrast, renal lesions were moderate to severe, and included prominent segmental tubular necrosis, granular and cellular casts, and mild interstitial non-suppurative inflammation. In the kidneys of animals examined later in the outbreak, there was early interstitial fibrosis as well as tubular regeneration, with numerous hyaline casts in the renal medulla. Inkweed plants had been heavily browsed, and recognisable portions of the plant were found in the gastrointestinal tracts of affected calves. Chemical analysis of inkweed material revealed triterpene saponins. No known hepatotoxic or nephrotoxic plants were identified in the paddocks. The hepatic lesions were not consistent with published descriptions of cyanobacterial toxicity. Sporidesmin toxicity was ruled out., Diagnosis: Hepatogenous photosensitivity, crystal-associated cholangiohepatopathy, toxic acute renal tubular necrosis, associated with the ingestion of P. octandra, and possibly complicated by cyanobacteria in the water., Clinical Relevance: Crystal-associated cholangiohepatopathy with photosensitivity in cattle is rare, and has only been reported in steroidal saponin-containing Brachiaria decumbens poisoning, in Brazil. The consistent pattern of toxic acute renal tubular necrosis was similar to that caused by the ingestion of Quercus, Amaranthus or Lantana spp. A combination of toxicities was conceivable but circumstantial evidence strongly implicated P. octandra. Further toxicological investigation of this plant is warranted before it can be listed as a known nephrotoxin of cattle.

Polyplex micelles formed with plasmid DNA (pDNA) and poly(ethylene glycol) (PEG)-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] exhibit effective endosomal escaping properties based on di-protonation of diamine side chains with decreasing pH, which improves their transfection efficiency and thus are promising candidates for local in vivo gene transfer. Here, PEG-PAsp(DET) polyplex micelles were further improved as in vivo systemic vectors by introduction of cholesterol (Chole) into the ω-terminus of PEG-PAsp(DET) to obtain PEG-PAsp(DET)-Chole. Introduction of the cholesterol resulted in enhanced association of block copolymers with pDNA, which led to increased stability in proteinous medium and also in the blood stream after systemic injection compared to PEG-PAsp(DET) micelles. The synergistic effect between enhanced polymer association with pDNA and increased micelle stability of PEG-PAsp(DET)-Chole polyplex micelles led to high in vitro gene transfer even at relatively low concentrations, due to efficient cellular uptake and effective endosomal escape of block copolymers and pDNA. Finally, PEG-PAsp(DET)-Chole micelles achieved significant suppression of tumor growth following intravenous injection into mice bearing a subcutaneous pancreatic tumor using therapeutic pDNA encoding an anti-angiogenic protein. These results suggest that PEG-PAsp(DET)-Chole micelles can be effective systemic gene vectors for treatment of solid tumors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 × 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution.

Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ∼60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 h. Hydrazones formed with the PMCA cross-linker showed no difference in release rates at pH 7.4 and 5.0.

Nucleic acid-based therapies offer great potential for treatment of a variety of diseases including cancer by modulating protein expression with DNA or small interfering RNA. However, realization of their full therapeutic potential is currently limited due to an inability to reach the target site in an active form. Identification of delivery barriers such as stability in circulation, resistance to degradation and entrapment in subcellular vesicles has led to development of sophisticated multifunctional synthetic polymers for forming ionic complexes with nucleic acids and also providing performance-enhancing features. The most promising designs comprise features to help increase stability in circulation and also contain functionality to aid in endosome escape of nucleic acid cargo after cellular internalization.

Dramatic advances in biological research have revealed the mechanisms underlying many diseases at the molecular level. However, conventional techniques may be inadequate for direct application of this new knowledge to medical treatments. Nanobiotechnology, which integrates biology with the rapidly growing field of nanotechnology, has great potential to overcome many technical problems and lead to the development of effective therapies. The use of nanobiotechnology in drug delivery systems (DDS) is attractive for advanced treatment of conditions such as cancer and genetic diseases. In this review paper for a special issue on biomaterial research in Japan, we discuss the development of DDS based on polymeric micelles mainly in our group for anti-cancer drug and gene delivery, and also address our challenges associated with developing polymeric micelles as super-functionalized nanodevices with intelligent performance.

Thiolated dimeric tetramethylrhodamine (TAMRA) was synthesized in a straightforward procedure utilizing commercially available 5(6)-succinimidyl TAMRA and cystamine hydrochloride. The thiol-containing TAMRA dimer displayed distinct spectral properties in reduced and oxidized forms; covalent dimer formation produced greater effects on the spectral properties than previously reported for noncovalent TAMRA dimers or dimers formed with shorter carbon spacers. The resulting TAMRA disulfide dimer exhibited a hypsochromic shift of 34 nm relative to the reduced monomer species and an isosbestic point at 532 nm between reduced monomeric and oxidized dimeric forms. Molar extinction coefficients of the monomer and dimer relative to moles of TAMRA were similar (6.61 x 10(4) M(-1) cm(-1) and 6.42 x 10(-4) M(-1) cm(-1), respectively). However, fluorescence emission was altered with >93% of dye fluorescence quenched in phosphate buffered saline upon dimer formation. A 520:554 nm absorbance intensity ratio of 2.64 was observed for the oxidized ssTAMRA dimer, almost twice as high as values reported for noncovalent TAMRA dimers. The resulting disulfide dye was easily reduced using both soluble and agarose gel immobilized tris(2-carboxyethyl) phosphine and fresh cell lysate from cultured RAW 264.7 macrophage cells. Absorbance intensity ratios at 554 and 520 nm were used to determine the oxidation half-life of a 1.2 x 10(-5) M solution of reduced TAMRA stored in ambient atmosphere to be approximately 50 h at 22 degrees C. The free thiol dye was further reacted with maleimide-derivatized poly(hydroxypropyl methacrylamide) to yield the dye-labeled polymer conjugate. This dye derivative should prove useful as a dithiol reduction-sensitive fluorescent probe in cellular tracking systems, as well as a thiol-based dye-labeling reagent due to its easy preparation from readily available materials, environmental sensitivity, and simple activation to produce distinct spectral states. The enhanced spectral properties of the covalent TAMRA dimer described here could be useful to prepare more advanced reporter molecules and bioconjugates.

A core-shell-type polyion complex (PIC) micelle with a disulfide cross-linked core was prepared through the assembly of iminothiolane-modified poly(ethylene glycol)-block-poly(L-lysine) [PEG-b-(PLL-IM)] and siRNA at a characteristic optimum mixing ratio. The PIC micelles showed a spherical shape of approximately 60 nm in diameter with a narrow distribution. The micellar structure was maintained at physiological ionic strength but was disrupted under reductive conditions because of the cleavage of disulfide cross-links, which is desirable for siRNA release in the intracellular reductive environment. Importantly, environment-responsive PIC micelles achieved 100-fold higher siRNA transfection efficacy compared with non-cross-linked PICs prepared from PEG-b-poly(L-lysine), which were not stable at physiological ionic strength. PICs formed with PEG-b-(PLL-IM) at nonoptimum ratios did not assemble into micellar structure and did not achieve gene silencing following siRNA transfection. These findings show the feasibility of core cross-linked PIC micelles as carriers for therapeutic siRNA and show that stable micellar structure is critical for effective siRNA delivery into target cells.

Critically-ill patients who have sustained multiple traumatic injuries have complex, and often conflicting, physiological needs. These have profound implications on the way in which nursing staff approach the physical positioning of these patients to minimize the risks of further physiological injury and damage, maintain homeostasis and promote optimum recovery. This article reviews and discusses the evidence base underpinning therapeutic positioning of the multiply-injured trauma patient within the intensive-care unit (ICU), focusing on patients with a known or suspected unstable spinal injury, pelvic injury, traumatic brain injury, chest injury, or multiple limb fractures. Included are guidelines on the therapeutic positioning of the multiply-injured trauma patient within the ICU, based on the current available evidence and also drawn from practical experience within the author's own place of work. There is also a brief discussion of how such guidelines may be introduced into clinical practice.

Photopolymerized poly(ethylene glycol) (PEG)-crosslinked hydrogels were assessed for their ability to serve as a payload vehicle to deliver a viable bacterial vaccine (Brucella abortus strain RB51 (RB51) to bison in Yellowstone National Park) ballistically using thermoplastic degradable Biobullets. PEG modified with degradable glycolide or lactide oligomers capped with photopolymerizable methacrylate groups served to crosslink the hydrogel vaccine carrier inside commercial hydroxypropylcellulose Biobullets. Release of 1 microm diameter model fluorescent particles from hydrogels followed known degradation trends for glycolide- and lactide-modified PEG hydrogels. All particles were released from PEG-co-glycolide hydrogels after approximately 10 days and PEG-co-lactide hydrogels after approximately 45 days following gel degradation. Minimal particle release was observed from pure PEG dimethacrylate hydrogels over 40 days. P. aeruginosa (strain PAO1) and RB51 live vaccines exhibit excellent viability following exposure to photopolymerization encapsulation within these gel matrices. Hydrogels photopolymerized into the payload chamber of Biobullets exhibit similar ballistic properties to commercially available Biobullets and penetrate and remain intact when fired intramuscularly into live elk for release of their gel payload in the host.

This study compared responses of bison calves to 10(10)CFU of Brucella abortus strain RB51 (SRB51) delivered by parenteral or ballistic methods. Two types of biobullet payloads were evaluated; compacted SRB51 pellets or SRB51 encapsulated in photopolymerized poly(ethylene glycol) hydrogels. Bison were vaccinated with saline, parenteral SRB51 alone, or in combination with Spirovac, or ballistically with compressed SRB51 or hydrogel biobullets. Bison parenterally vaccinated with SRB51 had greater (P<0.05) immunologic responses when compared to control bison. Co-administration of Spirovac as an adjuvant did not influence immunologic responses. As compared to compressed SRB51 biobullets, ballistic vaccination with hydrogel biobullets increased cellular immune responses at some sampling times. Our data suggest that hydrogel formulations of SRB51 may be a superior alternative to compressed SRB51 tablets for ballistic vaccination of bison. Although preliminary, data suggests that immunologic responses of bison to SRB51 hydrogel bullets are similar to responses after parenteral vaccination with SRB51.

Fluorescence in situ hybridization (FISH) analysis of the bone marrow of a 24-year-old man diagnosed with acute promyelocytic leukemia (APL) revealed a variant pattern with one fusion signal instead of the typical two fusions expected with the probe set used. The combined FISH and conventional chromosome analyses suggested that two subsequent translocations had occurred in this patient involving the same chromosomes 15 and 17. As the prognostic outcome in APL is strictly associated with the presence of a PML/RARA fusion, it is useful and necessary to perform both cytogenetic and FISH analyses of a variant t(15;17) to determine the status of the PML/RARA fusion.

This work describes the design and synthesis of a novel polymer conjugate to facilitate intracellular release of attached cargo. Water soluble poly[N(2-hydroxypropyl) methacrylamide] with derivatizable pendant side chains was used as the polymer carrier. The membrane active Tat peptide was masked with poly(ethylene glycol) to inhibit non-specific interactions with exterior cell membranes, and then covalently linked to poly[N-(2-hydroxypropyl) methacrylamide]. A novel heterobifunctional crosslinker was synthesized containing both maleimide and aldehyde functionality to allow attachment of peptides to the polymer backbone through a pH sensitive bond. Analysis of the proposed conjugate by gel permeation chromatography, nuclear absorbance spectroscopy, and absorbance measurements indicate that the desired compound has been synthesized.

Macromolecular therapeutics provide numerous benefits for the delivery of cytotoxic or poorly soluble drugs in vivo. However, these constructs often encounter barriers for drug delivery on both the systemic and subcellular level. Many soluble polymer carriers have been designed to surmount specific physiological barriers individually, but less work has been dedicated to designing an all-encompassing construct that addresses multiple therapeutic barriers at once. Incorporation of multiple agents already individually known to increase effectiveness into one carrier could further improve current drug delivery technology. Recent developments in subcellular delivery of therapeutic agents in soluble macromolecular carriers are discussed in the context of the future possibility for the design of an all-encompassing soluble multi-functional drug delivery vehicle.

Background: Survival rates from mechanical ventilation (MV) in allogeneic bone marrow transplantation are poor, but little is known about the need for and outcomes from MV in patients who undergo autologous hematopoietic stem cell transplantation (AHSCT)., Study Objective: To determine the frequency of and risk factors for the use of MV in recipients of AHSCT and to identify predictors of survival in mechanically ventilated AHSCT patients., Design: Retrospective, cohort analysis, Setting: Tertiary-care, university-affiliated medical center., Patients: One hundred fifty-nine consecutive patients who underwent AHSCT., Interventions: Patient surveillance and data collection., Measurements and Results: The primary outcome measure was the need for MV, and the secondary end point was survival after MV. Of 159 patients, 17 required MV (10. 7%). Three variables were associated with the need for MV: increasing age, use of total body irradiation in the conditioning regimen, and treatment with amphotericin B. As a screening test to predict the need for MV, no risk factor had a sensitivity or specificity > 82%. Three of the 17 mechanically ventilated patients (17.6%) survived to discharge. Only the mean APACHE (acute physiology and chronic health evaluation) II score separated survivors from nonsurvivors (21.7 vs 31.4; p = 0.029). Both the duration of MV and the length of stay in the ICU were similar in survivors and nonsurvivors., Conclusions: We conclude that MV is infrequently needed following AHSCT. Although survival after MV in these patients is limited, clinical variables do not reliably allow clinicians to prospectively identify patients destined to die.