James Z. Hui

Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities

Nanoparticle-based drug delivery systems have been developed to improve the efficacy and reduce the systemic toxicity of a wide range of drugs. Although clinically approved nanoparticles have consistently shown value in reducing drug toxicity, their use has not always translated into improved clinical outcomes. This has led to the development of “multifunctional” nanoparticles, where additional capabilities like targeting and image contrast enhancement are added to the nanoparticles. However, additional functionality means additional synthetic steps and costs, more convoluted behavior and effects in vivo, and also greater regulatory hurdles. The trade-off between additional functionality and complexity is the subject of ongoing debate and the focus of this Review.

Facile Method for the Site‐Specific, Covalent Attachment of Full‐Length IgG onto Nanoparticles

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry.

Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

Volume And Composition Of Reflux After Intravitreal Injection

Purpose: To quantify the amount of drug loss from cadaveric human eyes, which are injected via the pars plana with a known volume of dye at variable intraocular pressures. Methods: Eight cadaver eyes were divided into 2 intraocular pressure groups: normal (15 mmHg; 4 eyes) or high (30 mmHg; 4 eyes). Each eye was injected with 50 &mgr;L of hematoxylin dye, and the subsequent reflux was immediately collected on a Schirmer’s test strip. The test strip was scanned and digitally analyzed to determine the area of saturation and total color intensity present. Using a previously established equation, total volume of reflux and the amount of dye within that reflux were calculated. Results: The average total volume of refluxed fluid was 1.68 &mgr;L (median, 0.62 &mgr;L), with a range of 0 &mgr;L to 8.05 &mgr;L. The average volume of refluxed dye was 0.37 &mgr;L (median, 0.08 &mgr;L), with a range of 0 &mgr;L to 2.15 &mgr;L. On average, only 0.74% of the original 50 &mgr;L of injected dye was lost (median, 0.15%), with a range from 0% to 4.30%. Conclusion: Although the presence of subconjunctival bleb formation after intravitreal injection may be a concern to the clinician, data from the present study shows that only a very small amount of the injected therapeutic agent is lost in the reflux.

LASIC: Light Activated Site-Specific Conjugation of Native IgGs

Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

A Novel Method for the Measurement of Reflux from Intravitreal Injections: Data from 20 Porcine Eyes

Abstract Background: Reflux following intravitreal injection is a common phenomenon, but it is unknown how much, if any, medication is lost as a result. Reflux is known to be a combination of vitreous and the injected agent, but the relative composition is unknown. This article describes a novel method for the measurement of the volume and composition of reflux and presents data from porcine eyes. Methods: Twenty porcine eyes were injected with 0.05 ml of dye at intraocular pressures (IOPs) of 15, 20, 25 and 30 mmHg (five eyes per subgroup). Reflux was captured on filter paper and the area of saturation and color intensity of the dye were digitally analyzed. Total refluxed volume and proportion of dye versus vitreous fluid were calculated from linear regression lines created from known standards. Results: Average (median) total volume of reflux from all eyes was 1.19 μl (0.93 μl), volume of injected dye refluxed was 0.47 μl (0.11 μl) and composition of reflux was 20.8% dye (15.5%). Less than 1% of the injected dye was lost to reflux. There were no differences between IOP groups in the total volume refluxed, the total amount of dye refluxed, the average composition of the reflux or the amount of injected dye refluxed (df = 3 for all comparisons; p = 0.58, p = 0.51, p = 0.55, p = 0.51, respectively). Conclusions: This novel method allows for measurement of quantity and composition of reflux following intravitreal injection in vitro. While reflux occurs frequently, it is predominantly composed of vitreous, not the injected agent. In fact, <1% of the original injection was lost to reflux.

Simultaneous Quantification of Tumor Uptake for Targeted and Nontargeted Liposomes and Their Encapsulated Contents by ICPMS

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.

Site-specific antibody-liposome conjugation through copper-free click chemistry: a molecular biology approach for targeted photodynamic therapy(Conference Presentation)

Nanocarriers, such as liposomes, have the ability to potentiate photodynamic therapy (PDT) treatment regimens by the encapsulation of high payloads of photosensitizers and enhance their passive delivery to tumors through the enhanced permeability and retention effect. By conjugating targeting moieties to the surface of the liposomal nanoconstructs, cellular selectivity is imparted on them and PDT-based therapies can be performed with significantly higher dose tolerances, as off-target toxicity is simultaneously reduced.1 However, the maximal benefits of conventional targeted nanocarriers, including liposomes, are hindered by practical limitations including chemical instability, non-selective conjugation chemistry, poor control over ligand orientation, and loss of ligand functionality following conjugation, amongst others.2 We have developed a robust, physically and chemically stable liposomal nanoplatform containing benzoporphyrin derivative photosensitizer molecules within the phospholipid bilayer and an optimized surface density of strained cyclooctyne moieties for ‘click’ conjugation to azido-functionalized antibodies.3 The clinical chimeric anti-EGFR antibody Cetuximab is site-specifically photocrosslinked to a recombinant bioengineered that recognizes the antibody’s Fc region, containing a terminal azide.4 The copper-free click conjugation of the bioengineered Cetuximab derivative to the optimized photosensitizing liposome provides exceptional control over the antibody’s optimal orientation for cellular antigen binding. Importantly, the reaction occurs rapidly under physiological conditions, bioorthogonally (selectively in the presence of other biomolecules) and without the need for toxic copper catalysis.3 Such state-of-the-art conjugation strategies push the boundaries of targeted photodynamic therapy beyond the limitations of traditional chemical coupling techniques to produce more robust and effective targeted therapeutics with applications beyond conventional treatments.