Calcium phosphate/PLGA-mPEG hybrid porous nanospheres: A promising vector with ultrahigh gene loading and transfection efﬁciency Ke-Wei Wang, a Lin-Zhu Zhou, b Ying Sun, c Guo-Jun Wu, b Hong-Chen Gu, * b You-Rong Duan, * c Feng Chen a and Ying-Jie Zhu * a Received 24th August 2009, Accepted 12th November 2009 First published as an Advance Article on the web 15th December 2009 DOI: 10.1039/b917441a A facile room-temperature method for the fabrication of calcium phosphate (CaP)/PLGA-mPEG hybrid porous nanospheres has been developed. The hybrid CaP/PLGA-mPEG nanospheres obtained were 2060 nm in diameter and exhibited porous structure, and they were applied as DNA vectors for DNA loading and in vitro transfection. The results showed that the DNA binding capacity and transfection efﬁciency of the as-prepared hybrid porous nanospheres were much higher than those of the CaP precipitates prepared according to the standard CaP transfection procedure and mesoporous silica reported before. MTT assays conﬁrmed that the CaP/PLGA-mPEG hybrid porous nanospheres were quite safe. In addition, both CaP and PLGA-mPEG are biocompatible and biodegradable, thus the as-prepared CaP/PLGA-mPEG hybrid porous nanospheres are promising in gene delivery. Introduction Gene therapy is a promising therapeutic strategy to treat a variety of inherited and acquired diseases. This technique involves the delivery of genes to appropriate cells and their subsequent maintenance and expression. 1–3 Generally, gene delivery systems can be classiﬁed as viral and non-viral vectors. Although viral vectors possess high transfection efﬁciencies, they have potential safety problems, such as the possibility of recombination, strong immunogenicity, inﬂammatory response and carcinogenicity. 4–8 Non-viral vectors, including naked DNA injection, 9 electroporation, 10 gene gun, 11,12 liposomes, 13,14 cationic polymers 15–17 and inorganic nanoparticles, 18–21 show moderate transfection efﬁciencies, however, one of the signiﬁcant advantages is relatively safe compared with viral vectors. Among the non-viral systems, calcium phosphate (CaP)/DNA co-precipitate which was initially developed by Graham and van der Eb in 1973, 22 is one of the most frequently used methods for plasmid DNA (pDNA) transfer to mammalian cells in vitro. Owing to its excellent biocompatibility, biodegradability and adsorptive capacity for pDNA, 23 CaP precipitation has been widely adopted as a standard laboratory procedure for the delivery of oligonucleotides and pDNA during the past three decades. 24–29 However, the transfection efﬁciency is dependent on the experimental parameters, such as the concentration of CaCl 2 and DNA, reaction temperature, pH value, and the time between precipitation and transfection. 22,30 In addition, the uncontrollable rapid growth of CaP particles always leads to bulk precipitation and a heterogeneous size distribution, which induces low transfection efﬁciencies and large deviations in the transfection efﬁciency. Therefore, this technique suffers from the poor reproducibility and low transfection efﬁciency. To overcome these deﬁciencies, it is important to prevent the fast growth of CaP particles and avoid the formation of large-sized precipitates. Recently, considerable efforts have been made to develop nano-sized CaP gene vectors. For example, Kataoka et al. 31,32 prepared hybrid nanoparticles comprised of calcium phosphate, oligonucleotide (or pDNA) and a block copolymer of poly- (ethylene glycol)-block-poly(aspartic acid) (PEG-PAA), and PEG-PAA was used to modulate the size of CaP particles. Besides polymer-assisted methods, CaP/DNA nanoparticles were also obtained through microemulsion and emulsion approaches. 33,34 Although nano-sized CaP gene vectors have been successfully prepared by a variety of methods, there are still challenges for the preparation of CaP nanostructures with well deﬁned size and morphology. For example, in many cases the gene vectors are synthesized in the presence of DNA, thus the preparation experiments are limited to very mild conditions. And in some approaches, the surfactants are involved, which are difﬁcult to be removed completely, thus the safety problems arise. Herein, we report a facile room-temperature method for the preparation of CaP/PLGA-mPEG hybrid porous nanospheres in the presence of an amphiphilic block copolymer poly(DL-lac- tide-co-glycolide)-block-monomethoxy (polyethyleneglycol) (PLGA-mPEG). The as-prepared CaP/PLGA-mPEG hybrid porous nanospheres are explored as gene vectors for DNA loading and transfection. The experimental results show that the DNA binding capacity and transfection efﬁciency of CaP/ PLGA-mPEG hybrid porous nanospheres are much higher than the CaP precipitates prepared according to the standard proce- dure and mesoporous silica reported before. It is worth to emphasize that PEG is widely used as biocompatible polymers and the PLGA polymers currently possess US Food and Drug Administration (FDA) approval for use in a variety of a State Key Laboratory of High Performance Ceramics and Superﬁne Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China. E-mail: y.j.zhu@mail.sic.ac.cn b Med-X Institute, Shanghai Jiao Tong University, Shanghai, 200030, P. R. China. E-mail: hcgu@sjtu.edu.cn c Cancer Institute of Shanghai Jiao Tong University, Shanghai, 200032, P. R. China. E-mail: yrduan@shsci.org This journal is ª The Royal Society of Chemistry 2010 J. Mater. Chem., 2010, 20, 1161–1166 | 1161 PAPER www.rsc.org/materials | Journal of Materials Chemistry