Jianwei Du

Polypeptoids with tunable cloud point temperatures synthesized from N-substituted glycine N-thiocarboxyanhydrides

N-substituted glycine N-thiocarboxyanhydrides (NTAs) are alternative monomers for preparing polypeptoids. With an easy synthetic approach and stability during purification and storage, they have much more potential for mass production than the corresponding N-carboxyanhydrides (NCAs). Thermoresponsive copolypeptoids are synthesized by copolymerization of sarcosine NTA (Sar-NTA) with N-butylglycine NTA (NBG-NTA) initiated by benzylamine in THF at 60 °C. Polypeptoids with a degree of polymerization over 150 are obtained for the first time through primary amine-initiated NTA polymerizations. The molecular weights (MWs) and compositions of poly(sarcosine-r-N-butylglycine)s [P(Sar-r-NBG)s] are controlled by the feed molar ratios of [Sar]/[NBG]/[benzylamine]. The thermal behaviors of the copolypeptoids are investigated. Reactivity ratios of Sar-NTA and NBG-NTA are determined as 1.70(7) and 0.63(7), indicating a random distribution of the two monomers in the polypeptoid products. The structure and precise amino chain end of P(Sar-r-NBG) are confirmed by MALDI-ToF mass analysis. P(Sar-r-NBG)s have lower critical solution temperatures (LCST) and exhibit reversible phase transitions in aqueous solution. Their cloud point temperatures (Tcps) are tunable between 27 and 71 °C by adjusting the sarcosine molar fraction in copolymers. In addition, Tcp transitions depend on the MWs and the concentrations of the polypeptoids, as well as salt additives, to a certain degree. A biocompatibility study on P(Sar-r-NBG) reveals a controlled cytotoxicity related to the composition of polypeptoids. Easily accessible from NTA polymerizations, polypeptoids are therefore novel degradable materials with LCST for biomedical applications.

Cationized bovine serum albumin as gene carrier: Influence of specific secondary structure on DNA complexibility and gene transfection.

In this research, BSA, one of the natural rigid globular proteins with ca. 51% of α-helix secondary structure, was utilized to prepare cationized BSA (cBSA) as gene carrier. Tetraethylenepentamine (TEPA) or polyethylenimine (PEI1800) was grafted to BSA with different grafting levels. Based on the circular dichoism (CD) spectra, all cBSA remained α-helical structure to some degree. This was exciting to endow cBSA with quite different DNA complexibility and cellular biology behavior from the random coiled and flexible polycations such as PEI and poly-l-lysine (PLL). Strangely, the DNA condensability decreased with the increment of TEPA or PEI1800 grafting level. Also, the cBSA could condense DNA effectively to form irregular nanoparticles around 50-200nm above N/P ratio of 10. On account of the excellent hydration of BSA, the cBSA/DNA complexes revealed good colloidal stability under physiological salt condition. Cell culture experiments indicated this BSA-based gene carrier possessed good cellular compatibility. Surprisingly, cBSA/DNA complexes could be uptaken excellently by up to 90% cells. This might be owing to the agitation effect of α-helical structure and the positive potential of these complexes. BSA-PEI1800/DNA complexes with quick endosome escape even had transfection efficiency as high as PEI25k/DNA complexes. Overall, this paper provided us the potential of cBSA as gene carrier and might have some instructions in the design of protein-based gene delivery system.

Regulation the morphology of cationized gold nanoparticles for effective gene delivery

Recent research indicated that the morphology of nanoparticles could result in distinct biological behaviors, thus played an important role in designing efficient gene delivery systems. Among them, gold nanoparticles (AuNPs) with various shapes were widely studied due to the good biocompatibility and easy modification ability. Our recent research indicated that polyethyleneimine-g-bovine serum albumin (BSA-PEI) as non-viral gene vector showed good colloid stability and high transfection efficiency. In this work, BSA-PEI was utilized to modify gold nanospheres (AuNSs) and gold nanorods (AuNRs) to investigate the influence of the morphology on gene delivery. Both AuNS@BSA-PEI and AuNR@BSA-PEI nanoparticles condensed DNA effectively at N/P ratio above 5 and maintained spherical or rod-like morphology respectively. Due to the higher surface charge density at the tips, the rod-like gene complexes were prone to use the tips to contact with cell membrane, which facilitated to be uptaked by HepG2 cells. The endocytosis inhibition experiments showed some differences in the endocytic pathway. Gene transfection experiment showed that the rod-like complexes had almost 100-fold higher of transfection level than that of spherical complexes at the N/P ratio of 20. This work provided a potential strategy for further design of gene vectors with improved transfection results by adjusting the morphology of gene vectors.

Azo-capped polysarcosine-b-polylysine as polypeptide gene vector: A new strategy to improve stability and easy optimization via host-guest interaction.

Polypeptide has been extensively researched in gene/drug delivery system due to the good biocompatibility. Herein, we synthesized total-polypeptide copolymers, i.e. Azo(azobenzene)-capped polysarcosine-b-polylysine (ASL) with narrow molecular weight distribution by α-amino acid N-carboxyanhydride (NCA) polymerization. Although the molecular weight of PLL segment was only about 6 kDa, ASL could condense DNA effectively and form about 150 nm spherical nanoparticles at N/P ratio of 15. The surface charge was significantly reduced due to the shielding effect of polysarcosine (PSAR) shell. ASL/DNA PeptoPlexes showed good colloidal stability under physiological salt condition and complexation competition stability in the presence of counter polyanion, which might improve the circulation time in vivo. The tip design of azobenzene provided a facile way for ligand modification via host-guest interaction, which could be flexibly optimized by changing its functional tags responding to a request. Our data showed that the introduction of CD-R8 could promote the internalization of gene into cytoplasm and even nucleus owing to the membrane penetrating effect of R8. Cell culture experiments indicated as a total-polypeptide system, ASL showed good cellular viability and comparable gene transfection level as PLL with molecular weight of 50 kDa. Overall, PSAR served as an ideal alternative of PEG and this total-polypeptide system showed us a good direction for gene carrier design.

Intracellular fluorescent light-up bioprobes with different morphology for image-guided photothermal cancer therapy

Multifunctional nanoprobe was drawing increased attention in tumor diagnosis and therapy. The simple and effective establishment of the theranostic nanoplatforms was still under urgent need. Meanwhile, the targeting ability and morphology of nanoprobe were essential for the effective endocytosis, which could further affect the diagnosis. In this work, two morphologies of nanoprobes were fabricated using gold nanorods (AuNRs) and gold nanospheres (AuNSs). Thiolated-hyaluronic acid labeled with nile blue (HS-HA-NB), a near-infrared (NIR) fluorescence dye, was coated on the surface of the gold nanoparticles to form stable nanoprobes (AuNR@HS-HA-NB, AuNS@HS-HA-NB). The fluorescence of NB molecules quenched outside cells due to the fluorescence resonance energy transfer (FRET), and recovered after the HA degradation inside the cells. HA also could enhance cellular uptake in CD44 receptor highly expressed human breast carcinoma cells (MCF-7). In this way, bioprobes realized the MCF-7 cell images through intracellular fluorescent light-up. Comparing with the sphere bioprobe, the rod-shaped bioprobe dramatically promoted endocytosis to achieve a better diagnosis effect in a short time. After NIR light irradiation, severe MCF-7 apoptosis was observed with AuNR@HS-HA-NB existed. Our studies suggested that the AuNR@HS-HA-NB nanoparticles were the excellent candidates of versatile bioprobes to realize rapid, precise image and photothermal therapy to MCF-7 cells.

An easy gene assembling strategy for light-promoted transfection by combining host-guest interaction of cucurbit[7]uril and gold nanoparticles

Cucurbit[7]uril (CB[7]), a representative member of the host family cucurbit[n]uril, can host-guest interact with many guest molecules such as adamantane, viologen and naphthalene derivatives. This host-guest interaction provides an easy strategy in gene vector assembling. Furthermore, CB[7] can self-assemble on gold nanospheres (AuNSs). Herein, the combination of CB[7] and AuNSs provides both advantages of host-guest interaction and photo-thermal effect of AuNSs. In this study, polyethyleneimine (PEI) and polyethylene glycol (PEG) were separately interacted with CB[7] via host-guest interaction. Then by assembling on AuNSs, PEI and PEG were combined together to condense DNA into polyplexes as well as enhance circulation stability of the polyplexes. These gene vectors were found to have high cellular uptake efficiency and low cytotoxicity. Furthermore, the well distributed AuNSs in the polyplexes could transform light into heat under light exposure because of the photo-thermal effect. This was found to effectively promote the entry of gene into cytoplasm and highly enhanced gene transfection efficiency.

Polydopamine-based nanoparticles with excellent biocompatibility for photothermally enhanced gene delivery

For non-viral gene delivery systems, desirable endosomal release is crucial for the achievement of optimum therapeutic efficacy. In this work, polyethylenimine-modified polydopamine-based nanoparticles (PPNPs) with excellent biocompatibility were prepared. These PPNPs showed an average diameter of 13 nm with narrow size distribution. Besides, they could load pGL3 DNA effectively at a mass ratio of PPNPs to DNA above 5 and form complexes with spherical morphology (60–80 nm). And PPNPs/DNA complexes demonstrated good photothermal conversion ability. Due to the excellent biocompatibility of polydopamine, these PPNPs/DNA complexes showed low cytotoxicity to HepG2 cells, even after 15 minutes of NIR light irradiation. Furthermore, the PPNPs/DNA complexes with mass ratios of 23 and 30 showed higher transfection levels than Lipofectamine 2000. After exposing these complexes to near infrared (NIR) light with a power density of 2.6 W cm−2 for 15 min, the transfection level of PPNPs/DNA complexes tripled in HepG2 cells. The rise in gene transfection was attributed to the locally induced heat produced by the PPNPs/DNA complexes, which promoted endosomal membrane disruption and led to better endosomal escape. This result was also confirmed by confocal laser scanning microscope observation. Moreover, PPNPs/DNA complexes demonstrated excellent biocompatibility in hemolysis assays. At the mass ratio of 23 and DNA concentration of 20 μg mL−1, the hemolysis ratio of the PPNPs/DNA complexes was only 1%, lower than that of the PEI/DNA complexes. This PPNP nanocarrier was inspiring for the design of non-viral gene delivery systems with promoted therapeutic efficacy.

DNA-loaded microbubbles with crosslinked bovine serum albumin shells for ultrasound-promoted gene delivery and transfection

The microbubble is a kind of clinically applied ultrasound contrast agent in disease diagnosis that can also rupture under sonication to increase membrane permeability and promote gene entry into targeted cells. However, the development of ultrasound-mediated gene delivery might be restricted because genes and microbubbles were separated and would not reach the targeted cells simultaneously. Herein, a kind of crosslinked positive microbubbles (CPMBs) were prepared to load DNA as gene vectors to promote gene delivery efficiency. The BSA shell of the CPMBs was crosslinked with disulfide bonds, which obviously enhanced the stability of the CPMBs. Furthermore, the CPMBs revealed sonoporation effects comparable to those of clinically applied SonoVue microbubbles. As DNA and CPMBs were electrostatically linked as an entirety, they would reach cells simultaneously. Thus, with the aid of ultrasound, these DNA-loaded microbubbles promoted DNA entry into cytoplasm more effectively and obtained higher cellular uptake efficiency and better transfection efficiency than DNA-mixed microbubbles. Confocal microscopy results showed that rupturing of the CPMBs/DNA entire microbubbles under sonication could carry DNA directly into the cytoplasm or nucleus. All results indicated that the cytocompatible DNA-loaded microbubbles have promising prospects in ultrasound-mediated gene delivery.