==== Front TheranosticsTheranosticsthnoTheranostics1838-7640Ivyspring International Publisher Sydney 10.7150/thno.27815thnov08p3872EditorialDrug nanorods are potential new nanocarriers for intracellular protein delivery Gao Weiping ✉Sun Mengmeng Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China✉ Corresponding author: gaoweiping@tsinghua.edu.cnCompeting Interests: The authors have declared that no competing interest exists. 2018 21 6 2018 8 14 3872 3873 11 6 2018 12 6 2018 © Ivyspring International Publisher2018This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.Protein therapeutics are increasingly important for the treatment of many diseases; however, intracellular protein delivery remains a considerable challenge. To address this challenge, drug nanorods have emerged as new nanocarriers for enhanced intracellular protein delivery via bypassing endo-lysosomes, which was called a “drug-delivering-drug platform”. protein therapeuticsprotein deliverynanocarriernanomedicine ==== Body Protein therapeutics become popular in the treatment of human diseases including diabetes, cancer, infectious and inflammatory diseases, due to their high activity and high specificity as compared to small molecule drugs. Nevertheless, protein therapeutics suffer from poor stability, short circulatory half-life, poor-membrane permeability, and immunogenicity 1,2. To address these problems, many strategies have been developed for protein delivery, including polymer conjugation, long-circulating protein fusion, and nanocarriers such as nanogels, nanocapsules, virus-like particles, and cationic lipid nanoparticles. Indeed, dozens of PEGylated protein therapeutics, human serum albumin (HSA) fused protein therapeutics, and fragment crystallizable (Fc) fused protein therapeutics have been approved by the U.S. Food and Drug Administration (FDA) 3,4. However, current FDA-approved protein therapeutics aim at extracellular targets, not at intracellular ones. This is because it is tremendously difficult for therapeutic proteins to spontaneously enter cells and efficiently escape from the endo-lysosomal system into the cytosol to play roles 5-7. Although much effort has been directed to overcome these problems in intracellular protein delivery 8-10, it remains a significant challenge to efficiently deliver therapeutic proteins into the cytosol of cells. Recently, Xiaofei Xin et al. reported a drug-delivering-drug platform (HA-PNPplex) for intracellular delivery of protein and combined cancer treatment 11. In their work, rod-like pure drug nanoparticles (PNPs) of paclitaxel (PTX) stabilized with positively charged polymer polyethylenimine (PEI) were used as nanocarriers of proteins via electrostatic interactions between the positively-charged PNPs and the proteins to form PNPs/protein complexes (PNPplex). Subsequently, hyaluronic acid (HA) was coated onto these PNPplex to yield HA-PNPplex via electrostatic interactions between the PNPplex and HA. To prove the concept, caspase 3, the dominant mediator of apoptosis in mammalian cells 12,13, was chosen for the demonstration of therapeutic efficacy in a caspase-3-deficient MCF-7 tumor model. They found that HA-PNPplex delivered caspase 3 into cells through a non-lysosomal pathway, the caveolae-mediated internalization, which was determined by the rod-like shape, not by the surface modification. Consequently, HA-PNPplex raised caspase 3 level up to 6.5-fold in MCF cells. In a MCF-7 breast cancer mouse model, HA-PNPplex showed synergistic effect between the protein and PTX. The authors claimed that the platform provides a completely new strategy for protein therapies and delivery for other biomacromolecules, even for cancer immunotherapy. These data appear to be interesting for protein delivery; however, several issues need to be addressed in the future. First, PEI is believed to be toxic 14, although the authors claimed that HA-PNPplex had high biocompatibility and tolerability. We would suggest that biodegradable and biocompatible positively-charged polymers like polylysine should be selected instead of PEI. Second, many proteins are not negatively-charged or not negative enough to form stable complexes with positively-charged polymers through electrostatic interactions as mentioned in this work. Therefore, the authors should clarify this limitation in their paper. Third, it is not clear that how much HA could be adsorbed onto the surface of PNPplex and how stable HA-PNPplex was. Therefore, there are several subtle or uncertain factors to be considered in the preparation of HA-PNPplex, which may complicate the quality control of HA-PNPplex in manufacturing. To our knowledge, more and more nanomedicines were composed of more than two components like this work 15,16. The structures of the nanomedicines were usually not clear because it is very difficult to well characterize these nanostructures, especially in vivo. More attention should be paid to this problem from the viewpoint of translational medicine. Fourth, the tumor penetration of HA-PNPplex was not well studied in this work. A reasonable method to investigate tumor penetration is to measure the distance of protein migration from the vessel 17,18. Fifth, the authors claimed that PNPs would be useful to carry antigen proteins for cancer immunotherapy. One question is whether it is necessary or reasonable to use PNPs as nanocarriers for antigen delivery. The other question is whether PNPs are toxic to immune cells. In our opinion, this work provides new nanocarriers of PNPs for efficient intracellular delivery of some negatively-charged proteins via caveolae-mediated endocytosis and shows a synergy of PTX therapy and caspase 3 therapy. After solving the above problems, the nanocarriers of PNPs would be useful for combined cancer therapy. To date, many nanocarriers have been developed for intracellular protein delivery, but most of them are complicated in structures and functions along with insufficient characterization, which may result in poor reproducibility and further hinder their clinical translation. We believe that less is more, which is especially important for drug development. This study was financially supported by grants from the National Natural Science Foundation of China (21534006) and Beijing Natural Science Foundation (2172028). ==== Refs 1 Dowdy SF Overcoming cellular barriers for RNA therapeutics Nat Biotechnol 2017 35 222 9 28244992 2 Frokjaer S Otzen DE Protein drug stability: a formulation challenge Nat Rev Drug Discov 2005 4 298 306 15803194 3 Strohl W R Fusion proteins for half-life extension of biologics as a strategy to make biobetters BioDrugs 2015 29 215 239 26177629 4 Alconcel S N S Baas A S Maynard H D FDA-approved poly (ethylene glycol)-protein conjugate drugs Polym Chem 2011 2 1442 1448 5 Mout R Ray M Tay T Sasaki K Yesilbag Tonga G Rotello VM General strategy for direct cytosolic protein delivery via protein-nanoparticle co-engineering ACS Nano 2017 11 6416 21 28614657 6 Fu A Tang R Hardie J Farkas ME Rotello VM Promises and pitfalls of intracellular delivery of proteins Bioconjug Chem 2014 25 1602 8 25133522 7 Stewart MP Sharei A Ding X Sahay G Langer R Jensen KF In vitro and ex vivo strategies for intracellular delivery Nature 2016 538 183 92 27734871 8 Mitragotri S Burke PA Langer R Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies Nat Rev Drug Discov 2014 13 655 72 25103255 9 Gu Z Biswas A Zhao MX Tang Y Tailoring nanocarriers for intracellular protein delivery Chem Soc Rev 2011 40 3638 55 21566806 10 Yu M Wu J Shi J Farokhzad OC Nanotechnology for protein delivery: overview and perspectives J Control Release 2016 240 24 37 26458789 11 Xin X Teng C Du X Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route Theranostics 2018 8 13 3474 3489 30026860 12 Kothakota S Azuma T Reinhard C Klippel A Tang J Chu K Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis Science 1997 278 294 8 9323209 13 Wang Y Gao W Shi X Ding J Liu W He H Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin Nature 2017 547 99 103 28459430 14 Hong S Leroueil P R, Janus E K, Peters J L, Kober M M, Islam M T, et al. 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