synthetic parameters. It was found that the cell killing ability of DOX released from
any of the PEC types was higher than that of free DOX (as measured by flow
cytometry and CLSM). In oral carcinoma cells, a higher internalization of PEC
particles was found compared with lung carcinoma cells. The highest internalized
DOX amount was found for the crosslinked PEC particles (by capillary electrokinetic analysis). Among the various PEC formulations, the best was PEC with a
crosslinked shell. The authors emphasized the use of natural polysaccharides
because of safety concerns about empty PEC particles after drug release.
Interaction of PEC Particles with Endothelial Cells
A very concise study on the interaction between PEC particles and human microvascular endothelial cells (HMVEC) was reported by Hartig et al. [169]. A PEC
system with PEL components known from successful microencapsulation protocols
[170] was used. Cationic PEC particles were generated in highly nonstoichiometric
ratios from dosing a mixture of ALG and CHO as the molar minority component
into a mixture of fluorescein isothiocyanate (FITC)-modified poly(methylene-coguanidinium chloride) (PMCG) and spermine hydrochloride (SPM) as the molar
majority component. All PELs had rather low molecular weights, and CaCl 2 and a
Pluronic-type dispergator were additionally included. Consecutive centrifugation
was applied, resulting in particle diameters of around 160 nm and cationic surface
charge of around +35 mV. These PEC particles were incubated in cell culture
medium, resulting in slightly larger particle diameters and negative surface charge
(À10 mV). Cell proliferation studies and propidium iodide staining revealed
nontoxicity. Moreover, both the binding and internalization kinetics (2 h) of
fluorescent PEC (PMCG-FITC/SPM/ALG/CHO) particles were compared with
those of the free fluorescent FITC-PMCG at HMVEC (see Fig. 33a).
Anionic FITC-PEC particles showed saturation features with large amounts,
whereas free cationic FITC-PMCG showed linear behavior with small amounts in
both binding and internalization. CLSM imaging demonstrated perinuclear
accumulation (Fig. 33b), and related Z-sectioning revealed that PEC particles
were in the same plane as the cytoplasm. The authors argued that although the
surface charge of PEC particles was negative, positive patches of PMCG may bind
to polyanionic sites (proteoglycans) of the ECM of HMVEC, followed by
macropinocytosis as the internalization mechanism [169].
Huang et al. [126] reported studies on human umbilical vein endothelial cells
(HUVEC) cultures incubated with PEC particles of the systems CHT/DS, PEI/DS,
and PLL/DS, the charge signs of which were not reported in detail. Both VEGFloaded and VEGF-unloaded PEC particles (250 nm) were studied for their effects
on cell proliferation (mitogenic activity) by MTS (tetrazolium salt) assays.
Irrespective of the PEC composition, similar proliferation values were obtained at
the 4th day after incubation for all empty control PEC particle systems, for the free
VEGF, and for the controls. Only in the case of PEC-bound VEGF for all
polycation/polyanion systems was an enhanced HUVEC proliferation found.
Sizing, Shaping and Pharmaceutical Applications of Polyelectrolyte Complex. . .
247
any of the PEC types was higher than that of free DOX (as measured by flow
cytometry and CLSM). In oral carcinoma cells, a higher internalization of PEC
particles was found compared with lung carcinoma cells. The highest internalized
DOX amount was found for the crosslinked PEC particles (by capillary electrokinetic analysis). Among the various PEC formulations, the best was PEC with a
crosslinked shell. The authors emphasized the use of natural polysaccharides
because of safety concerns about empty PEC particles after drug release.
Interaction of PEC Particles with Endothelial Cells
A very concise study on the interaction between PEC particles and human microvascular endothelial cells (HMVEC) was reported by Hartig et al. [169]. A PEC
system with PEL components known from successful microencapsulation protocols
[170] was used. Cationic PEC particles were generated in highly nonstoichiometric
ratios from dosing a mixture of ALG and CHO as the molar minority component
into a mixture of fluorescein isothiocyanate (FITC)-modified poly(methylene-coguanidinium chloride) (PMCG) and spermine hydrochloride (SPM) as the molar
majority component. All PELs had rather low molecular weights, and CaCl 2 and a
Pluronic-type dispergator were additionally included. Consecutive centrifugation
was applied, resulting in particle diameters of around 160 nm and cationic surface
charge of around +35 mV. These PEC particles were incubated in cell culture
medium, resulting in slightly larger particle diameters and negative surface charge
(À10 mV). Cell proliferation studies and propidium iodide staining revealed
nontoxicity. Moreover, both the binding and internalization kinetics (2 h) of
fluorescent PEC (PMCG-FITC/SPM/ALG/CHO) particles were compared with
those of the free fluorescent FITC-PMCG at HMVEC (see Fig. 33a).
Anionic FITC-PEC particles showed saturation features with large amounts,
whereas free cationic FITC-PMCG showed linear behavior with small amounts in
both binding and internalization. CLSM imaging demonstrated perinuclear
accumulation (Fig. 33b), and related Z-sectioning revealed that PEC particles
were in the same plane as the cytoplasm. The authors argued that although the
surface charge of PEC particles was negative, positive patches of PMCG may bind
to polyanionic sites (proteoglycans) of the ECM of HMVEC, followed by
macropinocytosis as the internalization mechanism [169].
Huang et al. [126] reported studies on human umbilical vein endothelial cells
(HUVEC) cultures incubated with PEC particles of the systems CHT/DS, PEI/DS,
and PLL/DS, the charge signs of which were not reported in detail. Both VEGFloaded and VEGF-unloaded PEC particles (250 nm) were studied for their effects
on cell proliferation (mitogenic activity) by MTS (tetrazolium salt) assays.
Irrespective of the PEC composition, similar proliferation values were obtained at
the 4th day after incubation for all empty control PEC particle systems, for the free
VEGF, and for the controls. Only in the case of PEC-bound VEGF for all
polycation/polyanion systems was an enhanced HUVEC proliferation found.
Sizing, Shaping and Pharmaceutical Applications of Polyelectrolyte Complex. . .
247