A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery.

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.

The ability of this pH-sensitive reporter platform to rapidly screen ligands in nanoparticle format will enable identification and production of targeted NPs with desired lysosome trafficking properties.

Cyclopentadiene as a Multifunctional Reagent for Normal- and Inverse-Electron Demand Diels-Alder Bioconjugation.

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.

Metabolite-Based Modification of Poly(l-lysine) for Improved Gene Delivery.

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.

A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide.

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.

A Diene-Containing Noncanonical Amino Acid Enables Dual Functionality in Proteins: Rapid Diels-Alder Reaction with Maleimide or Proximity-Based Dimerization.

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.

Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers.

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.

A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

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.

Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.

Dual environment-responsive polyplex carriers for enhanced intracellular delivery of plasmid DNA.

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.

A Hoechst Reporter Enables Visualization of Drug Engagement In Vitro and In Vivo: Toward Safe and Effective Nanodrug Delivery.

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.

Effect of polymer structure on micelles formed between siRNA and cationic block copolymer comprising thiols and amidines.

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.

Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry.

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.

Peptides as a material platform for gene delivery: Emerging concepts and converging technologies.

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.

Optical properties and application of a reactive and bioreducible thiol-containing tetramethylrhodamine dimer.

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.

Environment-responsive block copolymer micelles with a disulfide cross-linked core for enhanced siRNA delivery.

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.

Preclinical evaluation of a GFRA1 targeted antibody-drug conjugate in breast cancer.

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.

Pyrrolobenzodiazepine Antibody-Drug Conjugates Designed for Stable Thiol Conjugation.

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 EC50 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.

Live RB51 vaccine lyophilized hydrogel formulations with increased shelf life for practical ballistic delivery.

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.

Vascular bursts enhance permeability of tumour blood vessels and improve nanoparticle delivery.

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.

Systemic delivery of siRNA by actively targeted polyion complex micelles for silencing the E6 and E7 human papillomavirus oncogenes.

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.