Nano-carrier for gene delivery and bioimaging based on carbon dots with PEI-passivation enhanced fluorescence
Introduction
The fusion of nanotechnology and medicine has led to the emergence of nanomedicine where a nanomaterial platform is used for gene/drug therapy and diagnostic probe [1], [2]. The central challenge in this field is to design multifunctional nano-carriers that combine both therapeutic and diagnostic capabilities. Recently, many kinds of inorganic nanomaterials, such as gold nanoparticles [3], [4], iron oxide nanoparticles [5], [6], silica nanoparticles [7], [8], semiconductor quantum dots [9], [10], [11], carbon nanotubes [12], [13], nanodiamonds [14] and graphene [15], have been explored as nonviral vector for nucleic acid delivery by chemical modifications on purpose.
Lately, photoluminescent carbon dots (C-dots), as one of new members of carbon nanomaterials family, have drawn tremendous attention in nanotechnology field [16]. These surface-passivated carbon nanoparticles, with diameters lower than 10 nm, have been utilized for the application of biolabeling owing to their remarkable advantages in stable photoluminescence (PL), broad excitation spectra, tunable emission wavelength, and excellent biocompatibility compared with conventional organic dyes and semiconductor quantum dots which have raised serious health and environmental hazard concerns [17]. Routes to construct C-dots can be generally classified into two groups: top-down and bottom-up methods. The top-down methods, by which C-dots are generally formed through post-treating carbon particles broken from a larger carbon structure, consist of arc discharge [18], laser ablation [19], [20], [21], electrochemical oxidation [22], [23], [24]. The bottom-up approaches comprise combustion [25], [26], thermal carbonization [27], acid dehydration [28] and ultrasonic treatment [29], by which C-dots are transformed from suitable molecular precursors. The approaches mentioned above always involve intricate processes, expensive original materials or great energy-consuming devices, and the yield of C-dots is very low. The synthesized C-dots, typically, are always oxidized by nitric acid and further surface-passivated by diamine-terminated organic molecule to gain obvious photoluminescence properties.
More recently, strong photoluminescent C-dots have been produced directly via microwave pyrolysis of carbohydrates solution in the presence of passivation agent, 4,7,10-trioxa-1, 13-tridecanediamine (TTDDA) using a domestic microwave oven in our lab [30]. The strong photoluminescent performance of C-dots, as we found, has a close connection with the introduction of N atoms onto the skeleton of carbon nanoparticles. Other studies also suggested that the N-containing diamine-terminated oligomeric poly-(ethylene glycol) (PEG1500N) played an important role in the surface passivation of C-dots [20], [28]. That was reminiscent of polyethylenimine (PEI), a commonly used high performance nonviral vector, consisting of much more amino groups in its molecular structure. So we hypothesize that, the use of PEI as the surface passivation agent will able to aid in generating the C-dots with both enhanced fluorescent properties and gene delivery ability. However, the traditional surface functionalization process of nanoparticles usually involves time-consuming multiple steps.
Previous investigations have mostly focused on the synthesis process, but the poor gene condensation capability has restricted the use of C-dots in gene delivery itself. Herein, we report a hybrid nano-carrier based on PEI-functionalized C-dots (CD-PEI) via one-step microwave assisted pyrolysis of inexpensive glycerol in the presence of branched PEI in a few minutes. The most favorable feature of this strategy is that the formation of carbon nanoparticles and the surface passivation with PEI can be realized simultaneously in one pot. It is expected that the outer cationic polymer layer has the ability to mediate plasmid DNA transfection, and the entrapped C-dots emitting discernible fluorescence can serve as bioimaging.
Section snippets
Materials and chemicals
Branched polyethylenimine (PEI) with molecular weight 25 kDa was purchased from Sigma–Aldrich (St Louis, MO, USA). Quinine sulfate (98%, suitable for fluorescence) was supplied by Fluka (New York, USA). Plasmid pGL3-control with SV40 promoter and enhancer sequences encoding luciferase (5262 bp) was obtained from Promega (Madison, WI, USA). 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl tetrazolium bromide (MTT, 98%) was supplied by Alfa-Aesar (Beijing, China). The plasmids were amplified in
Characterization of CD-PEI
In this work, we presented three PEI-passivated C-dots with different microwave irradiation time. In the following discussion, all the samples used for characterization were prepared under the conditions of 20 ml glycerol, 6 ml 10 mm phosphate, 0.5 g PEI25kD and microwave treatment for 10 min (CD-PEI-B) unless specifically mentioned. As shown in Fig. 1a, the prepared C-dots passivated with PEI exhibited excellent water-soluble properties and emitted blue luminescence under UV light (365 nm).
Conclusion
In summary, we successfully constructed a high-efficient dual functional nano gene vector based on PEI-passivated C-dots by one-step microwave assisted pyrolysis of glycerol in the presence of branched PEI25k. In this hybrid nano-carrier system, PEI played two crucial roles: surface passivation to endow the C-dots with strong photoluminescence, DNA condensation for gene transfection. The elaborately fabricated CD-PEI exhibited excellent water solubility and bright multicolor fluorescence with
Acknowledgement
The authors gratefully acknowledge the support for this work from the National Natural Science Foundation of China (Grant 50973082) and National Science and Technology Major Project of China (Grant 2012ZX10004801-003-007).
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