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For electrostatic LbL self-assembled shells, the surface charge of the outermost layer is important when interacting with cells. As described herein, 1 -micron polyelectrolyte shells with different surface charges and compositions were fabricated and in vitro interactions with the tumor cell line MCF-7 were studied using confocal laser scanning microscopy and flow cytometry. Shell surface charges were characterized by zeta-potential measurements. Polycation coated shells present positive surface charge prior to contacting serum-containing culture media, but surface charge became negative after one hour of culture media incubation.

Polyanion coated shells also displayed a surface charge change before and after incubation in serum-containing media. Among all surfaces, shells covered with lipid bilayers displayed the highest cell uptake percentage of A positive surface charge does not necessarily show a higher cell uptake than a surface with negative charges, and this may due to serum protein adsorption. To prevent protein adsorption, PEI25k-PEG5k copolymers ; ; were used as outermost layers for shell assembly. As demonstrated herein, polyelectrolyte shells with a copolymer coating PEI25k-PEG5k resulted in the least cell internalization These in vitro shell-cell interaction results may be useful to tailor the design of shell surfaces for particular applications in drug delivery and molecular sensing.

In certain embodiments, the surfaces of the nanoshells of the present invention may be modified by oligo- or poly-ethyleneglycol regions. This can be done by attaching oligo- or poly-ethylene glycols to the outermost surface of the subject nanoshells, e.

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The effect of particle surface chemistry plays an important role in the uptake process. As described herein, negatively charged proteins were adsorbed on PEI coated shells as indicated by the dramatic change of surface charge.

In such case, the amino group on PEI may not play a similar masking function as amidine groups. Compared to negatively charged particles, poly-L-lysine-modified microparticles were internalized under all conditions with highest efficiency, which is suggested to be mediated by their positive surface charge Thiele L et al Biomaterials 24 8 : But in other reports, particles with negatively charged surfaces are easier for cells to uptake compared to positively charged surfaces Heck JD et al Cancer Res In general, the neutral surface is considered the surface with the least cell uptake than positive or negative surfaces.

For example, as described herein, PEG coated particles such as particles coated with PEI25k-PEG5k have a much lower cell uptake compared to shells with other materials. Shell surface lipophilicity may also play an important role in cell uptake process. Lipid bilayers on shell surfaces presented the highest the negative charges and resulted in the highest particle uptake.

Similar results were also reported in other studies such as lipoprotein uptake, and it was suggested that the presence of lipoprotein lipase was very helpful to facilitate the uptake of lipoproteins Rinninger, F et al J Lipid Res 39 7 : As described herein, 1-micron shells are much bigger than lipoplex Ross, PC et al Gene Ther 6 4 and lipoprotein based drug delivery systems. The mechanism behind this high percentage of shell uptake is unknown but may be due to the lipophilic property that helps cell membrane fusion with lipid shells. The ability to incorporate lipid bilayers into polyelectrolyte shells is noteworthy since it represents a unique system with combined physical and chemical properties.

The special structure leads to function changes of which the shell membrane permeability is greatly reduced Moya, S et al Macromolecules It is important to combine the shell surface physical properties with other characteristics such as surface charge to determine how cells will respond to these microparticles.

It is known that the presence of albumin on particle surface usually decreases uptake by cells Moghimi, SM et al Biochim Biophys Acta ; Thiele, L Biomaterials 24 8 This maybe due to reduced opsonization of shells due to the dysopsonic activity of albumin. But different cells may react differently to albumin-coated particles.

For example, uptake experiments conducted with respiratory epithelium cells indicated that albumin-coated microspheres were neither bound nor internalized by the Calu- 3 cells but internalized by A cells as large as 3 micron Foster, KA et al 53 1 For the positively charged polymer shells, no direct relationship is presented between surface charge and particle uptake. PLL coated particles present the second lowest internalization of While high phagocytotic activities of macrophages and dendritic cells were observed in another study when microparticles 1 microns were coated with PLL instead of albumin Thiele, L et al.

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Thiele et al. As described herein, shells covered with PLL or albumin display similar uptake ratios of This may be because MCF-7 cells have different phagocytotic activity compared to macrophages and dendritic cells. As described herein, to ensure that shell-cell interactions are dependent on shell surface charge and other properties but not due to the cytotoxicity caused by shells, a three- day shell cytotoxicity study was conducted.

None of the shell formulations showed cell cytotoxicity in vitro as compared to the negative controls. To avoid the effects due to serum protein adsorption on shell surfaces, a parallel study was also carried out in order to observe shell behavior in a serum-free media. At the beginning of the study, original culture media was replaced with HBSS solution containing shells. A short time one hour incubation was performed as to prevent any side effects that may occur due to the absence of serum. In this situation, the flow cytometry data from in serum-containing media was analyzed.

As described herein, polyelectrolyte shells with different outermost layers were assembled and the shell-cell interactions were studied in vitro. A positive surface charge does not necessarily equal higher shell internalization. For shells with three different negative charges, higher cell internalization corresponds to a higher surface charge. Shell original surface charges were dramatically changed after one-hour incubation in serum-containing media.

Proteins such as albumin and globulin may be adsorbed onto polycations while other positively charged materials may interact with polyanions. The simplicity and versatility of LbL self-assembly provides a unique method to build hollow polyelectrolyte shells that may be used as drug carriers or fluorescence sensors. The data described herein may be useful when designing shell surface properties for different biomedical applications such as in cancer therapy. By modulating the surface charge of the nanoshells of the subject invention, the amount of nanoshells of the subject invention taken up by cells e.

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Cell Surface Engineering Fabrication of Functional Nanoshells

In certain embodiments, the surfaces of the nanoshells of the subject invention are modified by PEG. In certain further embodiments of the invention, PEG-modified shell surfaces reduce protein adsorption and may provide prolonged blood circulation for drug delivery applications. The size of a self-assembled polymer shell directly correlates with the core size.

In order to further reduce the particle size, the particles were subjected to a surface- erosion procedure. It has been reported that complete MF particle decomposition occurs after 20 seconds in a pH 1. Gao et al. By way of example, monodisperse 1. At different times, particle suspensions were analyzed by dynamic light scattering DLS 90Plus Submicron Particle Size Analyzer, Brookhaven Instruments to measure the particle diameters in solution at room temperature. Particle surface charge before and after treatment was also characterized by zeta-potential measurement in 1 mM KC1 solution.

Figure 3 A shows the particles' diameter as measured by DLS as a function of decomposition time at pH values 1. Treatment with pH 2. Decomposition kinetics at these pH values clearly deviates from the previous observation of linear size reduction over time at pH 1. At the higher pH values employed in the present invention, a dramatic particle size decrease in the first 20 minutes was observed in all of the HCl solutions.

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The data in Figure 3A demonstrate that particle diameter depends on both hydrolysis time and pH values. The acid- hydrolyzed, size-reduced MF particles are positively charged and have similar zeta-potential to the original larger particles. For example, the zeta-potential for the nm MF particles is Scanning electron microscopy SEM, Hitachi S model was used to further characterize the particle size and surface morphology.

At higher magnification x 25K , the original MF particles are shown to be spherical, with a smooth surface Panel D. Panels B and C demonstrate that the MF particles surprisingly maintained their spherical shape and monodisperse size distribution after 8 and 60 minute hydrolysis, respectively. Although rapid diffusion of acid into sub-micron particles of hydrated polymer might be expected, the relatively smooth surface morphology of the acid-hydrolyzed MF particles Panels E and F suggests that a surface-erosion process is occurring under these conditions.

Using the LbL self-assembly technique, a series of sub-nanometer polymer nanoshells with different diameters were fabricated, using the different sizes of MF particles prepared above as templates. Capsules composed of gelatin multilayers are known to effectively extend drug release half-life H. Ai, S. Jones, M. Villiers, Y. Lvov, J. Release 86 59 , and nanoshells comprising layers of gelatin represent a preferred embodiment of the present invention.

By way of example, MF particles were first suspended in 2 mg ml gelatin solution in pH 7.


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Centrifugation was used to remove excess polymer before coating the next layer. The process was repeated, until a total of five bilayers of gelatin and PDDA were introduced to the MF particle surface. Subsequently, core decomposition was carried out in a pH 1. It should be noted that choosing appropriate pH during assembly is important to maintain a sufficiently high degree of ionization for the charged polymers.

Gelatin is a protein-based zwitterionic molecule with an isoelectric point of 4. These nanoshells consisted of five alternating bilayers of gelatin and poly dimethyldiallyl ammomum chloride PDDA. Due to the fact that the inner MF cores were completely dissolved away, SEM images showed collapsed shell structure as a result of sample drying process.