Block copolymer micelles for drug delivery: design, characterization and biological significance

Engineered nanoparticles have the potential to revolutionize the diagnosis and treatment of many diseases; for example, by allowing the targeted delivery of a drug to particular subsets of cells. However, so far, such nanoparticles have not proved capable of surmounting all of the biological barriers required to achieve this goal. Nevertheless, advances in nanoparticle engineering, as well as advances in understanding the importance of nanoparticle characteristics such as size, shape and surface properties for biological interactions, are creating new opportunities for the development of nanoparticles for therapeutic applications. This Review focuses on recent progress important for the rational design of such nanoparticles and discusses the challenges to realizing the potential of nanoparticles.

Strategies in the design of nanoparticles for therapeutic applications

The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro- and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 μm by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.

https://www.pnas.org/content/pnas/105/33/11613.full.pdf

The effect of particle design on cellular internalization pathways

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure–function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.

Design and development of polymers for gene delivery

Azides, which are extremely rare in biological systems, are emerging as attractive chemical handles for bioconjugation.[1–5] In particular, the CuI catalyzed 1,3-dipolar cyclization of azides with terminal alkynes to give stable triazoles[6, 7] has been employed for the tagging of a variety of biomolecules,[8–12] activity-based protein profiling,[13] and the chemical synthesis of microarrays and small molecule libraries.[14]

Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions.

Self-assembling polycation/DNA complexes represent a promising synthetic vector for gene delivery. However, despite considerable versatility and transfectional activity in vitro, such materials are quickly eliminated from the bloodstream following intravenous injection (plasma α half-life typically less than 5 min). For targeted systemic delivery a more prolonged plasma circulation of the vector is essential. Here we have examined factors contributing to rapid elimination of poly(L-lysine) (pLL)/DNA complexes from the bloodstream, and implicate the binding of proteins to the polyelectrolyte complexes as a likely cause for their blood clearance. pLL/DNA complexes reisolated from serum associate with several proteins, depending on their net charge, although the major band on SDS-PAGE co-migrates with albumin. Serum albumin binds to pLL/DNA complexes in vitro, forming a ternary pLL/DNA/albumin complex which regains some ethidium bromide fluorescence and fails to move during agarose electrophoresis. Albumin also causes increased turbidity of complexes, and reduces their zeta potential to the same level (−16 mV) as is measured in serum. We propose that rapid plasma elimination of polycation/DNA complexes results from their binding serum albumin and other proteins, perhaps due to aggregation and phagocytic capture or accumulation of the ternary complexes in fine capillary beds.

Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery.

Cationic polymers can self-assemble with DNA to form polyelectrolyte complexes capable of gene delivery, although biocompatibility of the complexes is generally limited. Here we have used A-B type cationic-hydrophilic block co-polymers to introduce a protective surface hydrophilic shielding following oriented self-assembly with DNA. Block co-polymers of poly(ethylene glycol)–poly-l-lysine (pEG-pLL) and poly-N-(2-hydroxypropyl)methacrylamide–poly(trimethylammonioethyl methacrylate chloride) (pHPMA–pTMAEM) both show spontaneous formation of complexes with DNA. Surface charge measured by zeta potential is decreased compared with equivalent polycation–DNA complexes in each case. Atomic force microscopy shows that pHPMA–pTMAEM/DNA complexes are discrete spheres similar to those formed between DNA and simple polycations, whereas pEG–pLL/DNA complexes adopt an extended structure. Biological properties depend on the charge ratio of formation. At optimal charge ratio, pEG–pLL/DNA complexes show efficient ...

Characterization of Vectors for Gene Therapy Formed by Self-Assembly of DNA with Synthetic Block Co-Polymers

Phototriggering of the metal-free azide to acetylene cycloaddition reaction was achieved by masking the triple bond of dibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do not react with azides in the dark. Irradiation of cyclopropenones results in the efficient (Φ355 = 0.33) and clean regeneration of the corresponding dibenzocyclooctynes, which then undergo facile catalyst-free cycloadditions with azides to give corresponding triazoles under ambient conditions. In situ light activation of a cyclopropenone linked to biotin made it possible to label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. The cyclopropenone-based phototriggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner and may facilitate the preparation of microarrays.

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Selective Labeling of Living Cells by a Photo-Triggered Click Reaction

The overexpression of saccharides such as Globo-H, Lewis(Y) and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development have been complicated by difficulties in eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression.

/pdf/robust-immune-responses-elicited-by-a-fully-synthetic-three-58i6illjel.pdf

Margreet A. Wolfert

Papers

Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions.

Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery.

Characterization of Vectors for Gene Therapy Formed by Self-Assembly of DNA with Synthetic Block Co-Polymers

Selective Labeling of Living Cells by a Photo-Triggered Click Reaction

Robust immune responses elicited by a fully synthetic three-component vaccine