Evaluation of gene and protein delivery to cell via polymeric micelles

  

  

Evaluation of gene and protein delivery to cell via polymeric micelles



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1. ABSTRACT

 Purpose. Determine aortic endothelial cells permeation ability and mechanisms of the aqueous block copolymeric micelles, poly(ethylene oxide)-poly(benzyl aspartate) (PEO-PBLA) chemically conjugated with FITC by transport study and confocal laser scanning microscopy. Methods. The block copolymers’ PEO-PBLA-FITC was first synthesized and characterized by GPC and CMC, confocal microscopy. Permeation ability and mechanisms of polymeric micelles in aortic endothelial cells were evaluated by incubating with NaF, NaN ₃ , wortmannin, cytochalasin B inhibitors, at 20 °C, and under reverse condition. The extent of localization of uptake polymeric micelles was established by confocal microscopy. Results. The size of the aqueous PEO-PBLA-FITC polymeric micelles was detected around 56 nm with unimodal distribution by AFM. The CMC test revealed the fluorescence intensity increased to around 0.01~0.05 mg/ml. NaF, NaN ₃ , wortmannin, cytochalasin B inhibitors, at 20 °C, and under reverse condition inhibited the absorption of polymeric micelles through aortic endothelial cells with apparent permeability coefficients of 18.07±1.03, 12.98±0.93, 11.31±0.77, 12.44±1.23, 6.40±0.23, 11.11±0.46, 10.22±1.09x10

cm/sec, respectively.. Confocal laser microscopy showed that fluorescent compounds were distributed in the intracellular cytopolasm

and nucleus. Conclusion.

PEO-PBLA-FITC copolymeric micelles in an aqueous system were transported by energy dependent endocytosis and were localized on transcellular and nucleus endothelial cells.

Key words: Polymeric micelles, endocytosis, endothelial, FITC..

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2. INTRODUCTION



 The selective delivery of anticancer drugs to non-operable tumors via drug carriers is one approach to rational drug therapy(1-2). These drug delivery systems, including many different drug carriers are often directed attach epitopes present on tumor cells and carry drug which interfere with tumor cells. Usually, these maromolecular carriers have to cross the tumor blood vessel wall consisting of endothelial cells and a basement membrane which are a major barriers for delivery (3).

Polymeric micelles, one of several macromolecule delivery carriers, are made from adriamycin conjugated block copolymer PEO-PBLA for selective drug delivery and have a reported high in vivo anticancer activity against leukemia and solid tumors as well as form stable micelles in the present of serum and circulate in the blood stream for a long amount of time. In order to effectively utilize this character of the long circulation of polymeric micelles in the blood stream, understanding the transport ability of PEO-PBLA polymeric micelles in the aortic endothelial membrane may be essential. Thus, the purpose of the present study was to elucidate the nano range of polymeric micelles penetration mechanisms and their ability in bovine aortic endothelial cells by chemically using conjugated FITC with PEO-PBLA polymeric micelles. In addition, the study aimed at determining the localization of PEO-PBLA-FITC polymeric

micelles in the aortic endothelial cells by using confocal laser scanning microscopy. µ µ

3. RESULTS AND DISCUSSION

The conjugation, purification and micelle formation of PEO-PBLA-FITC in Hank’s buffer solution were similar to these in previous study (2). The total fluorescence intensity increases of a fluorescent probe upon micellization have been utilized to determine CMC for a host of surfactants. At low concentrations of PEO-PBLA-FITC (below 0.01 mg/ml), negligible changes in the total fluorescence intensity were observed. All the polymeric micelles solution permeation, and AFM and confocal microscopic measurements were carried out to at least above the CMC of the polymers.

The time transport profile of 0.5 mg/ml

PEO-PBLA-FITC polymeric micelles across

aortic endothelial monolayers in Hank’s

buffer solution was found about 4% after

120 minutes. Table 1 shows that 0.05-0.5

mg/ml polymeric micelles transport through

the aortic endothelial monolayer and

apparent permeability coefficients were

18.89 ±1.60, 15.05±1.90, and 18.07±1.03

x10 ^-7 cm/sec, respectively It was shown

that the transport properties were

statistically independent of the initial

concentration. Without endothelial

monolayers on cell culture insert, polymeric

micelles penetration rate increased around 10

times. Adding 5 mM NaF with polymeric

micelles to endothelial cells caused the

permeability to be inhibited to 12.98 ±0.93

x10 ^-7 cm/sec. In addition, NaN ₃ , induced a

similar reduction in the transport of

polymeric micelles in the case of NaF (table

1). Third, 100 nm wortmanin inhibited

32% permeation as well as reduced 65%

penetration by 0.1 mM chtochalasin B.

The uptake and influx of polymeric micelles in low temperature significantly decreased(P=11.31 ±0.77 x10 ^-7 cm/sec) as compared to the control group. On the other hand, in the reverse permeation studies, there was a marked asymmetry in the two unidirectional fluxes of polymeric micelles across the endothelial cells as shown in Table 1. However, the transport of polymeric micelles in the presence of both chambers of 10% FBS serum did not influence the apparent permeability coefficient (P). Finally, FITC only move through the PET membrane of cell culture insert and it was found to be similar to polymeric micelles through only the cell culture insert (table 1).

Table 1. Polymeric micelles PEO-PBLA-FITC transport on aortic endothelial monolayers.

App. Perm. Coeff. P ±±±± SEM (10

cm/sec)

Control 173.90 ±±±±7.07

0.05mg/mlPolymeric Micelles 18.89±±±±1.60 0.1mg/ml Polymeric Micelles 15.05±±±±1.90 0.5mg/ml Polymeric Micelles 18.07±±±±1.03 0.5 mg/ml+5 mM NaF 12.98±±±±0.93 0.5 mg/ml+5 mM NaN

11.31±±±±0.77

0.5mg/ml +100nM wortmannin 12.44±±±±1.23

0.5mg/ml+0.1mM cytochalasin B 6.40±±±±0.23 0.5mg/ml+20°°°°C temperature 11.11±±±±0.46 0.5 mg/ml+reverse effect 10.22±±±±1.09 0.5mg/ml +10% serum 18.75±±±±2.14

FITC only 202.50±±±±13.70

4. LOCALIZATION OF

PEO-PBLA-FITC POLYMERIC MICELLES ON AORTIC ENDOTHELIAL CELLS BY CONFOCAL LASER SCANNING MICROSCOPY

Fifteen minutes after incubation of 0.5 mg/ml PEO-PBLA-FITC polymeric micelles in confocal laser scanning microscopy, FITC (red color) was found to be distributed over the intracellular space area, primarily at

parts of endothelial monolayer cells with cytoplasm (Fig. 1a). The distributions were numerous and due to this, their close proximity, and their small particle size (around 50 nm), they were difficult to resolve individually in fluorescent microscopy. Simultaneous imaging of FITC and nuclear live stain Hoeschst 33342 (green color) showed the majority of FITC was located around the cytoplasm and nucleus (Fig. 1b), but not between adjacent intercellular spaces. In a vertical cross section FITC fluorescence could only be found distributed inside cells, but not on top of cells.

5.REFERENCES µ

1.Molema et al. Pharm. Res.. 14: 2(1997).

2.Yokoyama et al. Cancer Res. 50:

1693(1990)

3.Madea et al. Bioconjugate Chem. 3:

351(1992)

4.Liaw et al. Pharm. Res.. 15: 1721(1998).

a)

b)

Figure 1. Transport of 0.5 mg/ml

PEO-PBLA-FITC polymeric micelles

across the aortic endothelial cell

monolayer for 15 minutes by confocal

fluorescence microscopy. a) FITC

transport (red) were stained in the

cytoplasm and nucleus. Live

nucleus-staining by Hoeschst 33342

(green) was observed in the nucleus. b) a

closer look inside the cytoplasm, and

some FITC can also be observed in the

nucleus area.