US20150238534A1

US20150238534A1 - Novel hollow particles

Info

Publication number: US20150238534A1
Application number: US14/632,760
Authority: US
Inventors: Jui-Yang Lai; Li-Jyuan Luo; Zih-Han Hong; Kao-Yuan Chang; Po-Cheng Lin; Ming-Hsi Chuang
Original assignee: Gwo Xi Stem Cell Applied Technology Co Ltd
Current assignee: Gwo Xi Stem Cell Applied Technology Co Ltd
Priority date: 2014-02-27
Filing date: 2015-02-26
Publication date: 2015-08-27
Also published as: WO2015127900A1; US20150274923A1; CN104874025B; TW201533240A; TWI541272B; TWI671402B; US9815959B2; TW201533101A; WO2015127897A1; CN104874026B; CN104874025A; CN104874026A

Abstract

A hollow particle is provided. The hollow particle of the invention comprises (a) a hollow microparticle, (b) a plural of linking molecules binding the surface of the microparticle, (c) a polypeptide binding the linking molecule, and (d) a target molecule binding the linking molecule. The hollow microparticle of the invention can deliver more cells and provides a higher rate of cell transplantation by the hollow structures, polypeptides and target molecules. The present invention also provides a manufacture of the hollow microparticle and a cell carrier.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This Non-provisional application claims priority under 35 U.S.C. §119(a) on Patent Application No. 61/945,364 filed in American United Sates Feb. 27, 2014, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a particle, in particular to a hollow particle with polypeptide, which can be used as a cell culture equipment. The microparticle of the invention also can be used as a functional biomaterials system for tissue repair in vivo.

BACKGROUND OF THE INVENTION

Technology related to fundamental and applied tissue engineering has been advanced for the purpose of developing transplantable artificial tissues as part of regenerative medicine. Specifically, studies including stem cell proliferation and differentiation, development of cytocompatible and biocompatible three-dimensional scaffolds, and construction of a variety of tissue engineering tools are now the most active research areas in regenerative medicine. Among them, scaffolds that are used to deliver stem cells or tissue cells therein are critical for the development of artificial tissues and organs
Scaffold materials used for the regeneration of body tissues must act as a platform to which cells adhere to form three-dimensional tissues. They must also function as a temporary barrier between transplanted cells and host cells, and they must be nontoxic and biocompatible generating tolerable immune reactions, if any are to be generated. In addition, scaffold material must be biodegradable in vivo at a desired time when the transplanted cells have grown sufficiently to the point of being able to adequately function as a tissue.
Typically, scaffolds are prepared from synthetic or natural polymers or their composites, and are manufactured into three-dimensional structures which have a variety of morphologies and properties. Most commonly used synthetic biodegradable polymers include polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic acid-co-glycolic acid) (PLGA), poly-ε-caprolactone (PCL), and derivatives and copolymers thereof, which can be used as biomaterials for scaffold preparation. Naturally biodegradable polymers as exemplified by collagen, alginate, hyaluronic acid, gelatin, chitosan, fibrin, etc., are also very useful candidates for this purpose. A variety of different forms of materials, such as sponges, gels, fibers, and microbeads, are applied for the fabrication of scaffolds, and the most popular ones are porous sponges and injectable hydrogels.
Wang, M., et al (Tissue Engineering, Volume 16, Number 5, 2010) discloses a porous PLGA scaffold used for inducing the differentiation of adipose-derived stem cells (ADSCs) with a hepatic inducing medium. PLGA particles cannot significantly improve the proliferation of ADSCs in a general medium. The results indicate that ADSCs are difficult to adhesively growth on PLGA scaffolds.
Kim S E, et al (Colloids and Surfaces B: Biointerfaces 122 (2014) 457-464) discloses a porous PLGA particles with heparin-dopamine (Hep) and lactoferrin (LF) for inducing the proliferation and differentiation of cells. According to the analysis results, the cell proliferation is not significant even if the particles are modified.
Accordingly, there are many technical barriers in achieving the goal of tissue engineering perfectly. For instances, the space for cell culture is not enough, yield is too low, the amount of carried cells is too low, and success rate of cell transplantation is too low. Therefore, a functional biomaterials system is needed to be used as a cell culture scaffold and transplant carrier.

SUMMARY OF THE INVENTION

In view of the above-mentioned problems, the present invention provides a novel hollow microparticle, manufacture thereof, and a cell carrier containing the hollow microparticle of the invention. The hollow particles of the present invention have superior pore structures for cell culture, and can be linked with polypeptides and target molecules to improve the cell-loading properties.
The present invention provides a hollow particle comprising (a) a hollow microparticle, (b) a plural of linking molecules binding the surface of the hollow microparticle, (c) a polypeptide binding the linking molecule, and (d) a target molecule binding the linking molecule.
In one embodiment, the hollow particle has hollow structures.
In one embodiment, the material of the hollow particle is selected from a group consisting of polylactic acid, poly(butylene succinate), poly(butylene succinate-co-butylene adipate), poly(butylene adipate-co-terephthalate), polyglycolic acid, poly(lactic-co-glycolic acid), Polycaprolactone, polyvinyl alcohol, and the combination thereof.
In one embodiment, the linking molecule comprising an amino group, hydroxyl group, carboxyl group, or nitrile group.
In one embodiment, the polypeptide comprises IKVAV, RGD, YIGSR, REDV, DGEA, VGVAPG, GRGDS, LDV, RGDV, PDSGR, RYWLPR, LGTIPG, LAG, RGDS, RGDF, HHLGGALQAGDV, VTCG, SDGD, GREDVY, GRGDY, GRGDSP, VAPG, GGGGRGDSP, GGGGRGDY, FTLCFD, poly-lysine, or MAX-1.
In one embodiment, the target molecule comprises hyaluronic acid, oxidized hyaluronic acid, colleagen, glucocorticoid, galectin, or osteopontin.
The present invention also provides a method for producing the hollow particle, comprising (a) providing a hollow particle, (b) soaking the hollow particle in a amine solution to form amino groups on the surface of the hollow particle, (c) adding a polypeptide, wherein the polypeptide is bound to the amino group on the surface of the hollow particle, and (d) adding a target molecule, wherein the target molecule is bound to the amino group on the surface of the hollow particle.
The present invention further provides a cell carrier comprising the hollow particle of the invention and a cell.
A detailed description is given in the following embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The present invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:

FIG. 1 is a schematic diagram showing the structure of the hollow particle according to an embodiment of the invention.

FIG. 2 is a schematic diagram showing the steps involved in manufacturing an optical element of the present invention.

FIGS. 3 a-3 e illustrate the external and internal morphology of the hollow PLGA, PLGA-NH₂, PLGA-NH₂-IKVAV, PLGA-NH₂-IKVAV-oHA particles and solid PLGA-IKVAV particles.

FIG. 4 illustrates the suspension of the hollow particles of the invention and solid particles.

FIG. 5 illustrates the level of amino group on the particles of the invention.

FIG. 6 illustrates the IKVAV grafting level of the hollow particles of the invention. The particles have a IKVAV grafting level of 15 mmol/mg.

FIG. 7 illustrates the oHA grafting level of the hollow particles of the invention. The particles have a oHA grafting level of 10.5 mmole.

FIG. 8 illustrates the results of fourier transform infrared spectroscopy (FTIR) assay. PLGA, PLGA-NH2, PLGA-NH2-IKVAV, and PLGA-NH2-IKVAV-oHA particles have different functional groups, respectively.

FIGS. 9 a-9 d illustrate the results of Nuclear magnetic resonance (NMR) assay. PLGA, PLGA-NH2, PLGA-NH2-IKVAV, and PLGA-NH2-IKVAV-oHA particles have different functional groups, respectively.

FIGS. 10 a-10 b are microscopy images showing the ADSC cell adhesion.

FIG. 11 illustrates the cell numbers of ADSC cells cultured with different hollow particles.

FIGS. 12 a-12 b are microscopy images showing the growth of ADSC cells cultured with different hollow particles.

FIG. 13 illustrates the cell numbers of ADSC cell cultured with different hollow particles.

FIGS. 14 a-14 b are microscopy images showing the HepG2 cell adhesion.

FIG. 15 illustrates the cell numbers of HepG2 cell cultured with different hollow particles.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best-contemplated mode of carrying out the invention. This description is made for the purpose of illustrating the general principles of the invention and should not be taken in a limiting sense. The scope of the invention is best determined by reference to the appended claims.
FIGS. 1 and 2 illustrate embodiments of the hollow particle and manufacture thereof according to the invention. It should be understood that the drawings herein are made in simplicity, and are utilized for illustrating associated elements related to the invention. In practical usage however, the particle is more complexly structured.
In one aspect of the invention, the present invention provides a hollow particle comprising (a) a hollow microparticle, (b) a plural of linking molecules binding the surface of the hollow microparticle, (c) a polypeptide binding at least one molecule, and (d) a target molecule binding at least one linking molecule.
FIG. 1 simplified illustrates a first embodiment of a hollow microparticle of the invention. Referring to FIG. 1, hollow particle 100 comprises hollow microparticle 11.
As used herein, the term “hollow particle” refers to a particle comprising one or more polymer(s). The particle of the invention may vary widely, ranging from 50 to 180 μm, preferably 120 μm. The particle is sphere, and the shape and size are not limited.
The hollow particle is manufactured into three-dimensional structures which have a variety of morphologies and properties, such as cell culture and/or cell delivery. The three dimensional (3D) cell culture model can generate a biomimetic native three-dimensional microenvironment of the tissue to provide more space for cell culture and reduce the costs of cell culture and increase the cells yield.
As used herein, the term “cell” refers to any cell derived from a mammalian subject suitable for transplantation into the same or a different subject. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. The cell of the present invention preferably is a “stem cell”. The term “stem cell” refers to any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells. Stem cells emanate from all germinal layers (i.e., ectoderm, mesoderm, and endoderm). Non-limiting examples of stem cells include precursor cells for the neuronal, hepatic, nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar, cardiac, intestinal, or endothelial lineage.
Several types and configurations of nanoparticles are encompassed by the present invention and may be composed of a range of materials including, but not limited to, a biostable polymer, a biodegradable polymer, fullerenes, lipids, or a combination thereof. Biostable refers to polymers that are not degraded in vivo. Biodegradable refers to polymers that are capable of being disposed of after delivery to a disease locale in a patient, e.g., when exposed to bodily fluids such as blood, and can be gradually absorbed and/or eliminated by the body.
In one embodiment, the biodegradable polymer is selected from aliphatic polyesters, aliphatic copolyesters, or aliphatic and aromatic copolyesters; More preferably, the biodegradable polymer is selected from polylactic acid (PLA), poly(butylenes succinate) (PBS), poly(butylene succinate-co-butylene adipate) (PBSA), poly(butylene adipate-co-terephthalate) (PBAT), polyethylene glycolic acid (PGA), poly(lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL), and polyvinyl alcohol (PVOH) or combinations thereof.
Referring to FIG. 1, hollow microparticle 11 includes at least one linking molecules on the surface of hollow microparticle 11.
As used herein, the term “linking molecule” refers to a group comprising amino group (NH2), hydroxyl group (OH), carboxyl group (COOH), or nitrile group (CN), preferably, a amino group.
In one embodiment, in order to produce at least one amino group on the surface of the hollow microparticles, the hollow microparticles are soaked in a amine solution. The methods for producing amino groups are not limited, one skilled in the art would select a suitable method depended on the requirements of each individual application.
Referring to FIG. 1, linking molecule 13 is bound to polypeptide 15.
As used herein, the term “polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The polypeptide of the present invention refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. The polypeptide is from 2 to 20 amino acid residues in length, preferably from 2 to 10 amino acid residues. The polypeptides may comprise 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues. In one embodiment, the polypeptide comprises IKVAV, RGD, YIGSR, REDV, DGEA, VGVAPG, GRGDS, LDV, RGDV, PDSGR, RYWLPR, LGTIPG, LAG, RGDS, RGDF, HHLGGALQAGDV, VTCG, SDGD, GREDVY, GRGDY, GRGDSP, VAPG, GGGGRGDSP, GGGGRGDY, FTLCFD, Poly-Lysine, or MAX-1, preferably, IKVAV.
Referring to FIG. 1, linking molecule 13 is bound to target molecule 17.
As used herein, the term “targeting molecule” refers to any agent (e.g., peptide, protein, nucleic acid polymer, aptamer, or small molecule) that specifically binds to a target of interest. The target of interest may be a tissue, a cell type, a cellular structure (e.g., an organelle), a protein, a peptide, a polysaccharide, or a nucleic acid polymer (see, e.g., Cox and Ellington, Bioorg. Med. Chem. 9:2525-2531 (2001); Lee et al, Nuc. Acids Res. 32:D95-D100 (2004)). Aptamers can be selected which bind nucleic acid, proteins, small organic compounds, vitamins, inorganic compounds, cells, and even entire organisms.
Target molecule 17 includes, but is not limited to, an antibody, antigen-binding antibody fragment, bispecific antibody, affibody, diabody, minibody, ScFvs, aptamer, avimer, targeting peptide, somatostatin, bombesin, octreotide, RGD peptide, folate, folate analog or any other molecule known to bind to a disease-associated target. one skilled in the art would select a suitable target molecule depended on the requirements of each individual application.
The hollow particle 100 can be delivered to specific tissues or organs. In one embodiment, hollow particle 100 can be used to culture cells (e.g., stem cells) and as a support structure for transplantation in vivo. The hollow particle 100 comprises linking molecule 13. Because linking molecule 13 can be recognized by a specific cell receptor, the cell adhesion activity of hollow particle 100 can be improved. In addition, linking molecule 15 can be bound to the receptor proteins of specific tissues or organs to improve the carrier activity of hollow particle 100. Therefore, the present invention provides a cell carrier, composition or product for medical use, particularly used for tissue healing in liver fibrosis patients.
In another aspect of the invention, the present invention provides a method for producing a hollow particle, comprising (a) providing a hollow microparticle, (b) soaking the hollow microparticle in an amine solution to form amino groups on the surface of the hollow microparticle, (c) adding a polypeptide, and the polypeptide is bound to the amino groups on the surface of the hollow microparticle, and (d) adding a target molecule, and the target molecule is bound to the amino groups on the surface of the hollow microparticle. The detail manufacture processes are described in Example 1 of the invention.
Referring to FIG. 2 a, a hollow particle is provided. The hollow microparticle have a microspheres shape and a diameter of about 120 μm. The material of the hollow microparticle is biocompatible and biodegrades in vivo, and the hollow microparticle can be used for cell culture, and used as a carrier.
Referring to FIG. 2 b, the hollow microparticle is soaked in an amine solution to form amino groups on the surface of the hollow microparticle. The amine solution of the present invention comprises 1,6-hexanediamine/isopropanol solutions, Glycine solution, adipic dihydrazide (ADH) solution, PEG amine solution, NH2-PEG-NHS, or H2-PEG-NH2/DMSO solution. One skilled in the art would select an appropriate soaking time and condition depended upon different amine solutions.
Referring to FIG. 2 c, a polypeptide is added, and bound to the amino groups on the surface of the hollow microparticle. The hollow particle can be placed in a buffer solution to activate the amino groups on the surface of the hollow microparticle. The buffer solution of the present invention includes, but is not limited to, MES buffer, EDC, or NHS solution. The polypeptide then is added to form a bond between the polypeptide and the activated amino groups. The pH value of the buffer solution is between 2 and 10, preferably, 3 and 9, more preferably, 4 and 8. One skilled in the art would adjust pH value to activate amino groups depended upon the different buffer solution. The concentration of polypeptide is about 0.01 wt % to 50 wt % in the solution, preferably, 10 wt % to 30 wt %.
Referring to FIG. 2 d, a target molecule is added. The target molecule is bound to the unbound-amino groups on the surface of the hollow microparticle.
In one embodiment, the target molecules can self-reactive with amino groups. For example, hyaluronic acid (oHA) can be used as a target molecule, and the aldehyde groups of oHA can react with the amine groups of the hollow microparticles to form imine bonds.
In another embodiment, the target molecule is soaked in a buffer solution to activate the carboxyl groups of the target molecule. The hollow microparticle then is added to form bonds between the amino groups of the hollow microparticle and activated carboxyl groups.
The present invention also provides a cell carrier comprising the hollow particle of the invention and a cell.
As used herein, the term “cell” refers to any cell derived from a mammalian subject suitable for transplantation into the same or a different subject. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. The cell of the invention can be a somatic cell, artificial cell, or stem cell.
From the above, the hollow particle of the invention is capable of improving suspension cell culture to increase the yield of cells. Compared with traditional cell culture equipments and models, the hollow particle of the invention can be used to produce more cells at a lower cost.
The polypeptide of the hollow particle can be recognized by the specific cells to improve the cellular adhesion resulting in increasing the number of carried cells per hollow particle. Compared with traditional or commercial cell carriers, the hollow particle of the invention can carry more cells.
On the other hand, the hollow particle of the invention further comprises a target molecule which is bound to a desired target, such as specific tissues, organs, parts, or cells. In one embodiment, the target molecule is hyaluronic acid specifically bound to liver cells.
Therefore, the hollow particle of the invention can carry more cells by the hollow structures and polypeptides. Further, the hollow particle of the invention can be specifically bound to a desired target by the target molecules to deliver the cells to a target position. The cell transplantation efficacy is significantly increased by the hollow structures, polypeptides, and target molecules.

Embodiment 1

Manufacture of PLGA Hollow Particle

The PLGA particles were manufactured by double emulsion method. Briefly, 0.9 g of PLGA dissolved in 40 ml of methylene chloride (2.25%) was mixed with 20 ml of ddH2O, and then homogeneous mixed to conduct the first emulsification. 2.5 g of PVA and 250 ml of ddH2O were mixed to prepare 1% PVA solution. 1% PVA solution was cooled to 10° C., then mixed at 500 rpm. The product of the first emulsification was slowly dropped in the stirred 1% PVA solution by a dropper. After removal of the solution, the product was washed three times with ddH2O, and free-dried for three days to obtain PLGA particles. The PLGA particles were screened through screen mesh to obtain the PLGA particles with a diameter of 100 to 200 μm. Referring to FIG. 3 a, the scanning electron microscope (SEM) observation indicates that PLGA particles inside had porous structures.
1.1 Formation of Amino Groups on PLGA Particles
PLGA particles were soaked in 10% 1,6-hexanediamine/isopropanol solutions for 3 hours, and then centrifuged to obtain PLGA-NH2 particles. The supernatant was retained for Nihydrin assay described in Example 2 to detect the linkage of amino groups. The particles were washed three times with ddH2O. Amino groups were formed on the surface of PLGA particles to become PLGA-NH2 particles. Referring to FIG. 3 b, the surface of PLGA-NH2 was rough.
1.2 Grafting of PLGA-IKVAV
0.1 M of MES buffer solution (pH 5.5) was prepared. N,N-dimethylaminopropyl carbodiimide (EDC) and hydroxysuccinimide (NHS) were added in a ration of 1:1, and then IKVAV polypeptide was added to activate carboxyl groups for two hours. The ration of EDC, NHS and IKVAV was 5:5:1. The PLGA-NH2 was added for the grafting reaction to occur. The concentration of IKVAV was 14 wt % in the solution. After 24 hours, the PLGA-NH2-IKVAV particles were formed. The supernatant was retained for TNBS assay described in Example 3 to detect the Grafting of IKAVA. Referring to FIG. 3 c, IKVAV was covered on the surface of PLGA-NH2, and the surface of PLGA-NH2 was smooth.
1.3 Grafting of PLGA-oHA
Finally, 150 mg of hyaluronic acid (oHA) dissolved in ddH2O was slowly mixed with 99.5% of ethanol. 150 mg of PLGA-NH2-IKVAV particles were added and then mixed at 150 rpm for 24 hours at pH11. The supernatant was retained for TNBS assay to detect the grafting of oHA. The particles were washed two times with ddH2O, soaked in 30% and 95% ethanol for 10 minutes, respectively, and then freeze-dried. Because the aldehyde groups of oHA were reacted with the amine groups of the particles to form imine bonds, PLGA-NH2-IKVAV-oHA particles were produced, as shown in FIG. 3 d.
1.4 Suspension of PLGA Hollow Particle and PLGA Solid Particle
Suspended cells (5×105 cells/1 ml medium) and sterilized particles were added to a tube and observe the suspension of hollow and solid PLGA particles. Referring to FIG. 4, the hollow particles were uniformly suspended in the solution, and the solid particles were precipitated at the bottom of the tube.

Example 2

Assay of Amino Groups Grafting Level

Nihydrin assay was used to detect the grafting level of amino groups. PLGA-NH2 was reacted with ninhydrin, and then heated for 20 minutes. Ninhydrin was reacted with amino groups to form a violet solution, and the grafting level of PLGA-NH2 was detected by measuring the absorbance at a wavelength of 570 nm length using UV-visible. Referring to FIG. 4, the grafting level of amino groups was more than 50%.

Example 3

Assay of IKVAV Grafting Level

TNBS assay (ACS Chem. Neurosci., 2013, 4, 1229-1235) was used to detect the polymerization between amino groups of IKVAV and carboxyl group of PLGA-NH2. 1 ml of PLGA-NH2-IKVAV particles were reacted with 10 mM of t-buty carbazate for 24 hours at pH 9, then reacted with 2 ml of TNBS solution for 30 minutes. After reaction, 1 ml of the solution was mixed with 1 ml of 0.5 N HCl to measure the absorbance at a wavelength of 340 nm length. The absorbance values were compared before and after grafting to obtain the grafting level of IKVAV. Referring to FIG. 5, the grafting level of IKVAV was 11.5 mmol/mg.

Example 4

Assay of oHA Grafting Level

TNBS assay was used to detect the grafting level of oHA. 1 ml of particles were reacted with t-buty carbazate for 24 hours at pH 9, then reacted with 2 ml of TNBS for 30 minutes. After reaction, 1 ml of the solution was mixed with 1 ml of 0.5 N HCl to measure the absorbance at a wavelength of 340 nm length. The absorbance values were compared before and after grafting to obtain the grafting level of IKVAV. Referring to FIG. 6, the grafting level of IKAVA was 10.5 mmol/mg.

Example 5

Chemical Assay

Fourier transform infrared spectroscopy (FTIR) was used to detect the frequency of vibration or rotation of the PLGA, PLGA-NH2, PLGA-NH2-IKVAV, PLGA-NH2-IKVAV-oHA particles, and functional groups of materials were detected by infrared light to predict the grafting level of amino groups, oHA and IKVAV. Referring to FIG. 7, FTIR could determine the different functional groups in PLGA, PLGA-NH2, PLGA-NH2-IKVAV, and PLGA-NH2-IKVAV-oHA particles. The absorption peaks of the particles of the present invention could be determined at 1090, 1185, 1375, 1745, and 2943 cm-1, because the particles consisted of a polymer of PLA and PGA. In addition, the grafting of PLGA and NH2 would produce amide bounds so that the absorption peaks of amide bounds could be found at 1550, 1650, and 3358 cm−1. The results indicate PLGA having amino groups. Further, carboxyl groups also reacted with amino groups to form amide group in a process for IKVAV grafting. Thus, the significantly increased amide groups demonstrate the particles having IKVAV. Regarding oHA grafting, aldehyde groups of oHA were reacted with amino groups to form imine bonds so that the absorption peak of imine bonds could be found at 1614 cm−1. The carboxyl groups of oHA could be found at 1414 cm−1. The absorption peaks demonstrates indirectly the grafting of oHA.

TABLE 1

absorption peaks of functional groups identified by FTIR

Functional groups

C—O

CH₃

COOH

CH₂

Amide II

C═N

Amide I

C═O

CH

N—H

Peak (m⁻¹)	1090	1185	1375	1414	1450	1550	1614	1650	1745	2943	3358

Nuclear magnetic resonance (NMR) was used to detect the magnetic field absorb and re-emit electromagnetic radiation of the PLGA, PLGA-NH2, PLGA-NH2-IKVAV, PLGA-NH2-IKVAV-oHA particles, and functional groups of materials were detected by NMR spectroscopy to predict the grafting level of amino groups, oHA and IKVAV. Referring to FIG. 8, NMR could determine the different functional groups in PLGA, PLGA-NH2, PLGA-NH2-IKVAV, and PLGA-NH2-IKVAV-oHA particles. The peaks of the particles of the present invention could be determined at 1.5, 4.8 and 5.3 ppm, because the particles consisted of a polymer of PLA and PGA. In addition, the grafting of PLGA and NH2 would produce amide bounds so that the peaks of amide bounds could be found at 2.1 and 3.8 ppm. The results indicate PLGA having amino groups. Further, carboxyl groups also reacted with amino groups to form amide group in a process for IKVAV grafting. Thus, the significantly increased amide groups demonstrate the particles having IKVAV. Regarding oHA grafting, aldehyde groups of oHA were reacted with amino groups to form imine bonds so that the peak of imine bonds could be found at 8.5 ppm. The peaks demonstrates indirectly the grafting of oHA.

Example 6

Physical Assay

Using Zeta potential and Laser particle size analyzer to detect surface potential, particle size, voltage between dispersion and particle, scattering bars change in light scattering of PLGA, PLGA-NH2, PLGA-NH2-IKVAV, and PLGA-NH2-IKVAV-oHA for analyzing electrical surface charge distribution, average surface area, and mean size Refer to table. 2, Zeta potentiadetect PLGA, PLGA-NH2, PLGA-NH2-IKVAV, PLGA-NH2-IKVAV-oHA have different surface potential and electrical surface charge. The zeta potential of PLGA is −12.3 mv, but positive charge of amino base would neutralize the negative charge of PLGA after modifying amino base with peptide, and becomes neutral electrically. Because of IKVAV has positive charge residue of Lys and Arg, the electrical surface charge of particle modifying with IKVAV is about positive. And it is beneficial for increasing numbers of attaching cells on the particle. After at all, PLGA-IKVAV is modified with oHA, that aldehyde group of oHA is binding amino base and forming imine bond. Because of oHA has a carboxyl group residue, the electrical surface charge of PLGA-IKVAV-oHA is about 1.09 mv. According to the results, electrical surface charge of particle modified with IKVAV and oHA is about positive. It is not only beneficial for increasing numbers of attaching cells on the particle, but also receptor on the cell has specific interaction force. However, the experiment of cell attachment shows the difference between positive particle and neutral particle.
Electrical surface charge distribution, average surface area, and mean size are analyzed with Laser Diffraction Particle Size Analyzer. According to analyzing results, the mean size of particle is between 50 μm-200 μm, and the average size is about 120 μm. The particle is shaken in the response solution in the process of binding peptide and change the surface charge. The smaller particle would cluster large particle, and the larger particle would cleavage into small particle. So, the larger/smaller ratio of the each group of the particle is different. The surface of particle shows the surface for cell attaching. Because of the surface of particle is full of nano holes, the surface of particle is lower than 0.045 cm2/particle. The surface of PLGA-NH2 modified with amino base is rough, but the surface after modified with IKVAV and oHA becomes smooth, but the area of surface decrease.

TABLE 2

Zeta potential

Metrial	Average size	Average area

PLGA	120.05 μm	0.0371 cm²/per MS
PLGA-NH2	120.04 μm	0.0483 cm²/per MS
PLGA-IKVAV	120.03 μm	0.0391 cm²/per MS
PLGA-IKVAV-oHA	120.03 μm	0.0401 cm²/per MS

TABLE 3

Average size and area

	Material	Zeta potential

	PLGA	−12.3 mV
	PLGA-NH2	0.31 mV
	PLGA-IKVAV	3.03 mv
	PLGA-IKVAV-oHA	1.09 mv

Example 7

Growth of Cells

Adipose derived stem cells (ADSCs) were cultured in tissue culture polystyrene (TCPS), hollow PLGA, PLGA-NH2, and PLGA-NH2-IKVAV particles, and solid PLGA-NH2-IKVAV particles. After 24 hours, the qualitative and quantitative analyses were conducted by SEM microscopy, fluorescence microscopy, and WST-1 assay to determine the level of cell adhesion between different carriers and environments.
7.1 Safety Assay of Particles
ADSCs were cultured in a medium (2×105/group) for 4 hours until cell adhesion was completed. The different particles were added and co-cultured for 48 hours. Trypan blue was used to determine the number and survival rate of cells. The results indicate there were no difference between the different particles and control group (TCPS). Therefore, the different particles would not affect the growth of ADSC cells and the particle exhibited little or no toxicity to ADCS cells.

TABLE 4

Survival assay of ADSC cells attached on the particles

	Group	Cells number

	TCPS	9.23 × 10⁵
	PLGA	9.12 × 10⁵
	PLGA-NH₂	9.02 × 10⁵
	PLGA-IKVAV	9.17 × 10⁵
	PLGA-IKVAV-oHA	9.21 × 10⁵
	NC	0

7.2 ADSC Attached on Particles
The particles were added to a cell suspension solution (1×106 cells/1 ml medium) at 37° C. in a oven for cell adhesion and culture. The cell suspension solution containing particles was mixed every 10 minutes so that the cells were contact with and attached to particles. After cells attached to particles (about 4-6 hours), washed nonattached cells by PBS and fixed the cells by paraformaldehyde, and dehydrated by ethanol. Referring to FIG. 10 a, the number of cells adhering to PLGA or PLGA-NH2 was not many. Regarding to PLGA-IKVAV, most of cells were stacked on one part of the particles, because the particles were precipitated at the bottom of the tube. Conversely, after the hollow particles were modified with IKVAV or IKVAV-oHA, cells were attached on the surface of particles (cells were labeled by white arrow). Referring to FIG. 10 b, cells were stained with DAPI, and observed by fluorescence microscopy. The images of fluorescence microscopy are similar to that of SEM (cells were labeled by white arrow). FIG. 11 shows the results of quantitative analysis using WST-1. The results indicate that the amount of cells which was attached on the hollow particles modified with IKVAV was significantly increased.
7.3 Growth of Cells Attached on Particles
Particles were added to a cells suspension solution (1×106 cells/ml medium) at 37° C. in a oven for cell adhesion and culture. The cell suspension solution containing particles was mixed every 10 minutes so that the cells were contact with and attached to particles. After about 24 hours, the tube was shaken softly to increase the space and area available for cell culture. After about 72 hours, cells were adhered to the particles and capable of growth. After washed nonattached cells by PBS, the particles were fixed by paraformaldehyde, and dehydrated by ethanol. Cells were observed and analyzed by staining with DAPI and SEM microscopy. WST-1 assay and viable cell counts using Trypan blue were conducted. After 72 hours of cells adhesion to particles, the qualitative and quantitative analyses were conducted. FIG. 12 a shows the SEM images of cells cultured with PLGA, PLGA-NH2, PLGA-IKVAV, or PLGA-IKVAV-oHA. The particles of PLGA and PLGA-NH2 were coated with cells partially. The particles of solid PLGA-IKVAV were not exposed to cells and the cells were stacked at a same surface because the particles were precipitate at the bottom of the tube. Regarding PLGA-IKVAV and PLGA-IKVAV-oHA, the particles were fully and uniformly covered by cells (cells were labeled by white arrow). FIG. 12 b shows the fluorescence microscopy images of cells stained with DAPI. The images of fluorescence microscopy and SEM microscopy are similar (cells were labeled by white arrow). FIG. 13 shows the results of WST-1 assay. The results indicate that the amount of cells which was attached on the hollow particles modified with IKVAV was significantly increased.
Experimental 7 Fixed HepG2 Cells on the Particle
Culture HepG2 suspend medium (1×106 cells/1 ml) with particles in 37 o at the incubator. Each 10 minutes, shake slowly the cell-particle medium to make sure the cell attaching particles fully. After cell attaching on the particle (about 4 to 6 hours), take the particles out off the medium, and washes out the non-attaching cells with PBS solution. Then, fixed the cells with paraformaldehyde, and dehydrating cell and particles with alcohol. FIG. 14 a is staining with DAPI, and observed with fluorescence microscope, and it shows that the cells attached on the PLGA and PLGA-NH2 is numberless, but PLGA-IKVAV-oHA and PLGA-oHA can observe large number cells attaching on the particles averagely. FIG. 14 b shows the same result as FIG. 14 a. FIG. 15 is qualified with WST-1. Because of that, oHA and CD44 can increase large number cell attaching.
While the invention has been described by way of example and in terms of the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. To the contrary, it is intended to cover various modifications and similar arrangements (as would be apparent to those skilled in the art). Therefore, the scope of the appended claims should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements.

Claims

What is claimed is:

1. A hollow particle comprises

(a) a hollow microparticle,

(b) a plural of linking molecules binding the surface of the microparticle, and

(c) a polypeptide binding the linking molecule, and

(d) a target molecule binding the linking molecule.

2. The hollow particle according to claim 1, wherein the hollow particle hollow structures.

3. The hollow particle according to claim 1, wherein the material of the hollow particle is selected from a group consisting of polylactic acid, poly(butylene succinate), poly(butylene succinate-co-butylene adipate), poly(butylene adipate-co-terephthalate), polyglycolic acid, poly(lactic-co-glycolic acid), Polycaprolactone, polyvinyl alcohol, and the combination thereof.

4. The hollow particle according to claim 1, wherein the linking molecule comprises an amino group (NH₂), hydroxyl group (OH), carboxyl group (COOH), or nitrile group (CN).

5. The hollow particle according to claim 1, wherein the polypeptide comprises IKVAV, RGD, YIGSR, REDV, DGEA, VGVAPG, GRGDS, LDV, RGDV, PDSGR, RYWLPR, LGTIPG, LAG, RGDS, RGDF, HHLGGALQAGDV, VTCG, SDGD, GREDVY, GRGDY, GRGDSP, VAPG, GGGGRGDSP, GGGGRGDY, FTLCFD, Poly-Lysine, or MAX-1.

6. The hollow particle according to claim 1, wherein the target molecule comprises hyaluronic acid, oxidized hyaluronic acid, colleagen, glucocorticoid, galectin, or osteopontin.

7. A cell carrier comprises the hollow particle of claim 1 and a cell.

8. The cell carrier according to claim 7, wherein the cell is adhered to inner and/or surface of the hollow particle.