7 research outputs found
DNA Amplification in Neutral Liposomes for Safe and Efficient Gene Delivery
In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed <i>in vivo</i> gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application
Precise Targeting of Liver Tumor Using Glycol Chitosan Nanoparticles: Mechanisms, Key Factors, and Their Implications
Herein,
we elucidated the mechanisms and key factors for the tumor-targeting
ability of nanoparticles that presented high targeting efficiency
for liver tumor. We used several different nanoparticles with sizes
of 200–300 nm, including liposome nanoparticles (LNPs), polystyrene
nanoparticles (PNPs) and glycol chitosan-5β-cholanic acid nanoparticles
(CNPs). Their sizes are suitable for the enhanced permeation and retention
(EPR) effect in literature. Different <i>in vitro</i> characteristics,
such as the particle structure, stability, and bioinertness, were
carefully analyzed with and without serum proteins. Also, pH-dependent
tumor cell uptakes of nanoparticles were studied using fluorescence
microscopy. Importantly, CNPs had sufficient stability and bioinertness
to maintain their nanoparticle structure in the bloodstream, and they
also presented prolonged circulation time in the body (blood circulation
half-life <i>T</i><sub>1/2</sub> = about 12.2 h), compared
to the control nanoparticles. Finally, employing liver tumor bearing
mice, we also observed that CNPs had excellent liver tumor targeting
ability <i>in vivo</i>, while LNPs and PNPs demonstrated
lower tumor-targeting efficiency due to the nonspecific accumulation
in normal liver tissue. Liver tumor models were produced by laparotomy
and direct injection of HT29 tumor cells into the left lobe of the
liver of athymic nude mice. This study provides valuable information
concerning the key factors for the tumor-targeting ability of nanoparticles
such as stability, bioinertness, and rapid cellular uptake at targeted
tumor tissues
Chemical Tumor-Targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry
Tumor-targeting strategies for nanoparticles have been predominantly based on optimization of physical properties or conjugation with biological ligands. However, their tumor-targeting abilities remain limited and insufficient. Furthermore, traditional biological binding molecules have intrinsic limitations originating from the limited amount of cellular receptors and the heterogeneity of tumor cells. Our two-step <i>in vivo</i> tumor-targeting strategy for nanoparticles is based on metabolic glycoengineering and click chemistry. First, an intravenous injection of precursor-loaded glycol chitosan nanoparticles generates azide groups on tumor tissue specifically by the enhanced permeation and retention (EPR) effect followed by metabolic glycoengineering. These ‘receptor-like’ chemical groups then enhance the tumor-targeting ability of drug-containing nanoparticles by copper-free click chemistry <i>in vivo</i> during a second intravenous injection. The advantage of this protocol over traditional binding molecules is that there are significantly more binding molecules on the surface of most tumor cells regardless of cell type. The subsequent enhanced tumor-targeting ability can significantly enhance the cancer therapeutic efficacy in animal studies
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>
Facile Method To Radiolabel Glycol Chitosan Nanoparticles with <sup>64</sup>Cu via Copper-Free Click Chemistry for MicroPET Imaging
An efficient and straightforward
method for radiolabeling nanoparticles
is urgently needed to understand the <i>in vivo</i> biodistribution
of nanoparticles. Herein, we investigated a facile and highly efficient
strategy to prepare radiolabeled glycol chitosan nanoparticles with <sup>64</sup>Cu via a strain-promoted azide–alkyne cycloaddition
strategy, which is often referred to as click chemistry. First, the
azide (N<sub>3</sub>) group, which allows for the preparation of radiolabeled
nanoparticles by copper-free click chemistry, was incorporated to
glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne
derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA) chelator, was synthesized for preparing the preradiolabeled
alkyne complex with <sup>64</sup>Cu radionuclide. Following incubation
with the <sup>64</sup>Cu-radiolabeled DBCO complex (DBCO-PEG<sub>4</sub>-Lys-DOTA-<sup>64</sup>Cu with high specific activity, 18.5 GBq/μmol),
the azide-functionalized CNPs were radiolabeled successfully with <sup>64</sup>Cu, with a high radiolabeling efficiency and a high radiolabeling
yield (>98%). Importantly, the radiolabeling of CNPs by copper-free
click chemistry was accomplished within 30 min, with great efficiency
in aqueous conditions. In addition, we found that the <sup>64</sup>Cu-radiolabeled CNPs (<sup>64</sup>Cu-CNPs) did not show any significant
effect on the physicochemical properties, such as size, zeta potential,
or spherical morphology. After <sup>64</sup>Cu-CNPs were intravenously
administered to tumor-bearing mice, the real-time, <i>in vivo</i> biodistribution and tumor-targeting ability of <sup>64</sup>Cu-CNPs
were quantitatively evaluated by microPET images of tumor-bearing
mice. These results demonstrate the benefit of copper-free click chemistry
as a facile, preradiolabeling approach to conveniently radiolabel
nanoparticles for evaluating the real-time <i>in vivo</i> biodistribution of nanoparticles