Mono-amino acid linkers enable highly potent small molecule-drug conjugates by conditional release.

The endosome cleavable linkers have been widely employed by antibody-drug conjugates and small molecule-drug conjugates (SMDCs) to control the accurate release of payloads. An effective linker should provide stability in systemic circulation but efficient payload release at its targeted tumor sites. This conflicting requirement always leads to linker design with increasing structural complexity. Balance of the effectiveness and structural complexity presents a linker design challenge. Here, we explored the possibility of mono-amino acid as so far the simplest cleavable linker (X-linker) for SMDC-based auristatin delivery. Within a diverse set of X-linkers, the SMDCs differed widely in bioactivity, with one (Asn-linker) having significantly improved potency (IC50 = 0.1 nM) and fast response to endosomal cathepsin B cleavage. Notably, this SMDC, once grafted with effector protein fragment crystallizable (Fc), demonstrated a profound in vivo therapeutic effect in aspects of targetability, circulation half-life (t1/2 = 73 h), stability, and anti-tumor efficacy. On the basis of these results, we believe that this mono-amino acid linker, together with the new SMDC-Fc scaffold, has significant potential in targeted delivery application.

Development of a spermine lipid for transient antibody expression.

Transient expression is the only way to quickly obtain a small scale of antibodies for biomedical research and therapeutic evaluation. The agents for transfecting the suspension cells, e.g. PEI or commercial agents, either lack efficiency or excessively expensive. Herein, a novel spermine-based lipid was developed and fabricated into a cationic liposome for antibody expression. This new transfection agent, designated as sperminoliposome, is feasible, cheap, and highly effective to produce antibodies. Compared to PEI, a 3 times higher yield of antibody was obtained by sperminoliposome during the transient expression of cetuximab in suspension 293F cells. Characterizations confirmed that the expressed antibody is fully functional and eligible for further research. Our study provides an effective tool for the rapid production of antibodies economically and feasibly.

In vivo therapeutic effects of small molecule-drug conjugates enhanced by Fc grafting.

Small molecule-drug conjugate (SMDC) shows great potential as a new class of targeted chemotherapeutic agents to tackle cancer. However, its in vivo therapeutic effect is compromised by its poor pharmacokinetic parameters. Herein we describe an approach that enables the precise conjugation of SMDC on N-terminus of the Fc protein to produce a SMDC-Fc bioconjugate (Fc1070) with superior specificity, affinity and potency to tumor cells. In vivo, Fc1070 exhibited an antibody-like pharmacokinetic profile with a long circulation half-life (t1/2 = 79 h) and pro-liver clearance pathway, that is distinct from the parent SMDC (t1/2 = 0.5 h and renal clearance). Intravenous injection of Fc1070 can eliminate the tumor with a single dosing of 7 mg/kg. Coupled with a predefined ligand toolbox, this approach allows the fast generation of other SMDC bioconjugates on demand, thus extending the format easily to other tumor targets. This may provide a general approach for the development of SMDC with enhanced therapeutic properties.

Cytosolic delivery of proteins by cholesterol tagging.

Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Conventional protein delivery techniques fail to address this fundamental problem in that protein cargos are predominantly delivered inside cells via endocytosis, a remarkably effective cell defense mechanism developed by Mother Nature to prevent intact biomolecules from entering the cytoplasm. Here, we report a unique concept, noncovalent cholesterol tagging, enabling virtually any compact proteins to permeate through the cell membrane, completely bypassing endocytosis. This simple plug-and-play platform greatly expands the biological target space and has the potential to transform basic biology studies and drug discovery.

Insight into the siRNA transmembrane delivery-From cholesterol conjugating to tagging.

Small interfering RNA (siRNA), combining the features of unprecedented potency, target-specificity, and the unique sequence-based disease-intervention model, has received immense considerations over the past decades in the academia and pharmaceutical industry. siRNA fits the criteria of being drug-likely enough to meet with the therapeutic purpose, but its clinical translation has been impeded for a long time by the poor efficiency of in vivo delivery. To reach the cytosol where the RNA interference (RNAi) takes place, siRNA delivery faces a serial of systemic and cellular barriers, especially the endosomal sequestration that would prevent the majority of siRNA from cytosol entry. Transmembrane delivery of siRNA represents a new avenue for efficient delivery by bypassing the endosomal pathway. This rationale is bolstered by the high efficiency of viral entry by membrane fusion, but rarely pursued by artificial siRNA delivery systems. Here, this article provides an opinion of transmembrane delivery by hydrophobic modulation of siRNA. We give a brief introduction of the current siRNA delivery modes, including the hydrophobic cholesterol siRNA conjugates. The cholesterol tagging technology is design on the rationale of hydrophobic siRNAs approach, but hydrophobic modulation throughout the whole siRNA backbone for efficient membrane fusion and transmembrane delivery. The challenge and potential of this technology for preclinical development are also discussed. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Chemical modulation of siRNA lipophilicity for efficient delivery.

siRNA holds tremendous promise to knockdown the disease-promoting gene in any cell type that it can be delivered. The biggest challenge facing the clinical translation of siRNA is to overcome the barriers that impede the in vivo delivery. siRNA is a highly hydrophilic macromolecule with poor pharmacological properties. The critical requirement for broadening the systemic siRNA delivery is to confer on siRNA the 'drug-like' properties such as the lipophilicity. In comparison with unmodified siRNA, lipophilic siRNA has a better cell-membrane compatibility and permeability. The lipophilic siRNA can also hijack the lipid trafficking pathway and natural drug carrier albumin to achieve an advanced systemic delivery, with properties including the tissue targeting, long circulation half-life, the improved pharmacokinetic profile, bioavailability and in vivo safety. In this review, we summarized the recent progress on lipophilic siRNA development, in particular about the chemical methods to manipulate siRNA lipophilicity. The covalent and noncovalent methodologies, including backbone modification, lipid conjugation, chelation method and others, are discussed. We place particular emphasis on the most clinically advanced method, the lipid siRNA conjugation. The application of lipophilic siRNA on nanoparticle delivery system is also covered.

Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery.

Studies on siRNA delivery have seen intense growth in the past decades since siRNA has emerged as a new class of gene therapeutics for the treatment of various diseases. siRNA bioconjugate, as one of the major delivery strategies, offers the potential to enhance and broaden pharmacological properties of siRNA, while minimizing the heterogeneity and stability-correlated toxicology. This review summarizes the recent developments of siRNA bioconjugate, including the conjugation with antibody, peptide, aptamer, small chemical, lipidoid, cell-penetrating peptide polymer, and nanoparticle. These siRNA bioconjugate, either administrated alone or formulated with other agents, could significantly improve pharmacokinetic behavior, enhance the biological half-life, and increase the targetability while maintaining sufficient gene silencing activity, with a concomitant improvement of the therapeutic outcomes and diminishment of adverse effects. This review emphasizes the delivery application of these siRNA bioconjugates, especially the conjugation strategy that control the integrity, stability and release of siRNA bioconjugates. The limitations conferred by these conjugation strategies have also been covered.

Noncovalent tagging of siRNA with steroids for transmembrane delivery.

Short interfering RNA (siRNA) has broad applications in biology and medicine, and holds tremendous potential to become a new class of therapeutics for many diseases. As a highly anionic macrobiomolecule, its cytosolic delivery, however, has been a major roadblock in translation. Here, we report the development of small, bifunctional chemical tags capable of transporting siRNA directly into the cytosol. The bifunctional tags consist of a siRNA-binding moiety that interacts with siRNA non-covalently, and a steroid domain that readily fuses with the mammalian cell membrane. In contrast to the conventional covalently conjugated siRNA-steroid that enters cells largely via endocytosis which substantially limits siRNA bioavailability, the non-covalently tagged siRNA is cell membrane-permeant, avoiding the endocytic pathway. This new methodology enables effective RNA interference (RNAi) without the need of cationic transfection or endosomolytic agents, opening a new avenue for intracellular delivery of native biologics.

A ribonucleoprotein octamer for targeted siRNA delivery.

Hurdles in cell-specific delivery of small interfering RNA (siRNA) in vivo hinder the clinical translation of RNA interference (RNAi). A fundamental problem concerns conflicting requirements for the design of the delivery vehicles: cationic materials facilitate cargo condensation and endosomolysis, yet hinder in vivo targeting and colloidal stability. Here, we describe a self-assembled, compact (~30 nm) and biocompatible ribonucleoprotein-octamer nanoparticle that achieves endosomal destabilization and targeted delivery. The protein octamer consists of a poly(ethylene glycol) scaffold, a sterically masked endosomolytic peptide and a double-stranded RNA-binding domain, providing a discrete number of siRNA loading sites and a high siRNA payload (>30 wt%), and offering flexibility in both siRNA and targeting-ligand selection. We show that a ribonucleoprotein octamer against the polo-like kinase 1 gene and bearing a ligand that binds to prostate-specific membrane antigen leads to efficient gene silencing in prostate tumour cells in vitro and when intravenously injected in mouse models of prostate cancer. The octamer's versatile nanocarrier design should offer opportunities for the clinical translation of therapies based on intracellularly acting biologics.

Publisher Correction: A ribonucleoprotein octamer for targeted siRNA delivery.

In the version of this Article originally published, in Fig. 3b, middle row, the units 'nM' were incorrect and should have been 'min'. And, in Fig. 4f, in the bottom row, the data in the middle and right panels were mistakenly duplicated from the panels above. These errors have now been corrected.

Dramatic enhancement of the detection limits of bioassays via ultrafast deposition of polydopamine.

The ability to detect biomarkers with ultrahigh sensitivity radically transformed biology and disease diagnosis. However, owing to incompatibilities with infrastructure in current biological and medical laboratories, recent innovations in analytical technology have not received broad adoption. Here, we report a simple, universal 'add-on' technology (dubbed EASE) that can be directly plugged into the routine practices of current research and clinical laboratories and that converts the ordinary sensitivities of common bioassays to extraordinary ones. The assay relies on the bioconjugation capabilities and ultrafast and localized deposition of polydopamine at the target site, which permit a large number of reporter molecules to be captured and lead to detection-sensitivity enhancements exceeding 3 orders of magnitude. The application of EASE in the enzyme-linked-immunosorbent-assay-based detection of the HIV antigen in blood from patients leads to a sensitivity lower than 3 fg ml-1. We also show that EASE allows for the direct visualization, in tissues, of the Zika virus and of low-abundance biomarkers related to neurological diseases and cancer immunotherapy.

Functional peptides for siRNA delivery.

siRNA is considered as a potent therapeutic agent because of its high specificity and efficiency in suppressing genes that are overexpressed during disease development. For nearly two decades, a significant amount of efforts has been dedicated to bringing the siRNA technology into clinical uses. However, only limited success has been achieved to date, largely due to the lack of a cell type-specific, safe, and efficient delivery technology to carry siRNA into the target cells' cytosol where RNA interference takes place. Among the emerging candidate nanocarriers for siRNA delivery, peptides have gained popularity because of their structural and functional diversity. A variety of peptides have been discovered for their ability to translocate siRNA into living cells via different mechanisms such as direct penetration through the cellular membrane, endocytosis-mediated cell entry followed by endosomolysis, and receptor-mediated uptake. This review is focused on the multiple roles played by peptides in siRNA delivery, such as membrane penetration, endosome disruption, targeting, as well as the combination of these functionalities.

Functional Photoacoustic Imaging of Gastric Acid Secretion Using pH-Responsive Polyaniline Nanoprobes.

A stomach functional imaging technique based on photoacoustics achieves noninvasive gastric acid secretory assessment utilizing pH-responsive polyaniline nanoprobes. A testing protocol mimicking clinical practice is established using a mouse model. After imaging, the nanoprobes are excreted outside the body without inducing systematic toxicity. Further optimization and translation of this technology can help alleviate patients' suffering and side effects.

Cross-Platform DNA Encoding for Single-Cell Imaging of Gene Expression.

Integration of imaging data across different molecular target types can provide in-depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target-type-specific labeling methods. We show that cross-platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.

Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization.

Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core-shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing.

Self-folded redox/acid dual-responsive nanocarriers for anticancer drug delivery.

Self-folded redox/acid dual-responsive nanocarriers (RAD-NCs) are developed for physiologically triggered delivery of anticancer drugs. The evidenced redox/acid responsiveness, facile decoration of ligands, and active tumor-targeting capability of RAD-NCs suggest their potential as a promising formulation for tumor-targeted chemotherapy.

Clickable protein nanocapsules for targeted delivery of recombinant p53 protein.

Encapsulating anticancer protein therapeutics in nanocarriers is an attractive option to minimize active drug destruction, increase local accumulation at the disease site, and decrease side effects to other tissues. Tumor-specific ligands can further facilitate targeting the nanocarriers to tumor cells and reduce nonspecific cellular internalization. Rationally designed non-covalent protein nanocapsules incorporating copper-free "click chemistry" moieties, polyethylene glycol (PEG) units, redox-sensitive cross-linker, and tumor-specific targeting ligands were synthesized to selectively deliver intracellular protein therapeutics into tumor cells via receptor-mediated endocytosis. These nanocapsules can be conjugated to different targeting ligands of choice, such as anti-Her2 antibody single-chain variable fragment (scFv) and luteinizing hormone releasing hormone (LHRH) peptide, resulting in specific and efficient accumulation within tumor cells overexpressing corresponding receptors. LHRH-conjugated nanocapsules selectively delivered recombinant human tumor suppressor protein p53 and its tumor-selective supervariant into targeted tumor cells, which led to reactivation of p53-mediated apoptosis. Our results validate a general approach for targeted protein delivery into tumor cells using cellular-responsive nanocarriers, opening up new opportunities for the development of intracellular protein-based anticancer treatment.

Bio-inspired synthetic nanovesicles for glucose-responsive release of insulin.

A new glucose-responsive formulation for self-regulated insulin delivery was constructed by packing insulin, glucose-specific enzymes into pH-sensitive polymersome-based nanovesicles assembled by a diblock copolymer. Glucose can passively transport across the bilayer membrane of the nanovesicle and be oxidized into gluconic acid by glucose oxidase, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH sensitive nanovesicle that in turn triggers the release of insulin in a glucose responsive fashion. In vitro studies validated that the release of insulin from nanovesicle was effectively correlated with the external glucose concentration. In vivo experiments, in which diabetic mice were subcutaneously administered with the nanovesicles, demonstrate that a single injection of the developed nanovesicle facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 5 days.

Folding graft copolymer with pendant drug segments for co-delivery of anticancer drugs.

A graft copolymer with pendant drug segments can fold into nanostructures in a protein folding-like manner. The graft copolymer is constructed by directly polymerizing γ-camptothecin-glutamate N-carboxyanhydride (Glu(CPT)-NCA) on multiple sites of poly(ethylene glycol) (PEG)-based main chain via the ring open polymerization (ROP). The "purely" conjugated anticancer agent camptothecin (CPT) is hydrophobic and serves as the principal driving force during the folding process. When exposed to water, the obtained copolymer, together with doxorubicin (Dox), another anticancer agent, can fold into monodispersed nanocarriers (with a diameter of around 50 nm) for dual-drug delivery. Equipped with a PEG shell, the nanocarriers displayed good stability and can be internalized by a variety of cancer cell lines via the lipid raft and clathrin-mediated endocytotic pathway without premature leakage, which showed a high synergetic activity of CPT and Dox toward various cancer cells. In vivo study validated that the nanocarriers exhibited strong accumulation in tumor sites and showed a prominent anticancer activity against the lung cancer xenograft mice model compared with free drugs.

ATP-triggered anticancer drug delivery.

Stimuli-triggered drug delivery systems have been increasingly used to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. Here we utilize adenosine-5'-triphosphate (ATP) as a trigger for the controlled release of anticancer drugs. We demonstrate that polymeric nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment. The half-maximal inhibitory concentration of ATP-responsive nanovehicles is 0.24 µM in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.