U.S. patent application number 16/113449 was filed with the patent office on 2018-12-20 for biodegradable scaffolds.
This patent application is currently assigned to Board of Regents of the University of Texas System. The applicant listed for this patent is Board of Regents of the University of Texas System. Invention is credited to Rachel Buchanan, Mauro Ferrari, Christine Smid, Ennio Tasciotti.
Application Number | 20180361030 16/113449 |
Document ID | / |
Family ID | 45441480 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180361030 |
Kind Code |
A1 |
Ferrari; Mauro ; et
al. |
December 20, 2018 |
BIODEGRADABLE SCAFFOLDS
Abstract
In some embodiments, the present invention provides compositions
that comprise: (1) a biodegradable polymer matrix; and (2) at least
one biodegradable reinforcing particle that is dispersed in the
matrix. In some embodiments, the biodegradable reinforcing particle
is selected from the group consisting of porous oxide particles and
porous semiconductor particles. In additional embodiments, the
compositions of the present invention further comprise a (3)
porogen particle that is also dispersed in the matrix. In further
embodiments, the compositions of the present invention are also
associated with one or more active agents. In various embodiments,
the active agents are associated with the biodegradable polymer
matrix, the biodegradable reinforcing particle, and/or the porogen
particle. In various embodiments, the compositions of the present
invention may be utilized as scaffolds, such as scaffolds for
treating bone defects. Further embodiments of the present invention
pertain to methods of making the compositions of the present
invention.
Inventors: |
Ferrari; Mauro; (Houston,
TX) ; Buchanan; Rachel; (Austin, TX) ; Smid;
Christine; (Austin, TX) ; Tasciotti; Ennio;
(Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents of the University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
Board of Regents of the University
of Texas System
Austin
TX
|
Family ID: |
45441480 |
Appl. No.: |
16/113449 |
Filed: |
August 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15270324 |
Sep 20, 2016 |
10058633 |
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16113449 |
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13809291 |
Mar 22, 2013 |
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PCT/US2011/029832 |
Mar 24, 2011 |
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15270324 |
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61363126 |
Jul 9, 2010 |
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61363835 |
Jul 13, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/56 20130101;
A61L 2300/406 20130101; A61L 2300/64 20130101; A61L 2300/252
20130101; A61L 2300/41 20130101; A61L 27/3847 20130101; A61L 27/16
20130101; A61L 27/20 20130101; A61L 27/3834 20130101; A61L 27/58
20130101; A61L 2430/02 20130101; A61L 2300/44 20130101; A61L 27/54
20130101; A61L 27/446 20130101; A61L 27/18 20130101 |
International
Class: |
A61L 27/56 20060101
A61L027/56; A61L 27/18 20060101 A61L027/18; A61L 27/58 20060101
A61L027/58; A61L 27/44 20060101 A61L027/44; A61L 27/38 20060101
A61L027/38; A61L 27/54 20060101 A61L027/54; A61L 27/16 20060101
A61L027/16; A61L 27/20 20060101 A61L027/20 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. W911NF-09-1-0044, awarded by the U.S. Department of Defense.
The government has certain rights in the invention.
Claims
1. A composition comprising: (a) a biodegradable matrix comprised
of at least one unsaturated polymer; and (b) at least one
biodegradable reinforcing particle dispersed in the matrix, wherein
the at least one biodegradable reinforcing particle is selected
from the group consisting of porous oxide particles and porous
semiconductor particles.
2. The composition of claim 1, wherein the biodegradable matrix
comprises an unsaturated biodegradable polymer.
3. The composition of claim 2, wherein the at least one unsaturated
polymer comprises poly(propylene fumarate) (PPF).
4. The composition of claim 1, further comprising hydrogel porogen
particles, dispersed within the biodegradable matrix.
5. The composition of claim 4, wherein the hydrogel porogen
particles comprise at least one of alginates, fibrins, gelatins,
and poly(lactic-co-glycolic acid) (PLGA).
6. The composition of claim 1, further comprising at least one of
biocompatible vesicles, liposomes, and micelles.
7. The composition of claim 1, further comprising at least one
active agent.
8. The composition of claim 7, wherein the at least one active
agent is selected from the group consisting of therapeutics,
imaging agents, anti-inflammatory agents, antibiotics, proteins,
platelet rich plasma, stem cells, degradation inducers of porous
particles, and combinations thereof.
9. The composition of claim 4, wherein the hydrogel porogen
particles further comprise at least one biodegradable porous
particle.
10. The composition of claim 9, wherein the at least one
biodegradable porous particle is a silicon porous particle.
11. The composition of claim 9, wherein a surface of the at least
one biodegradable porous particle is modified with a biodegradable
polymer.
12. The composition of claim 11, wherein the biodegradable polymer
comprises agarose, a hydrogel, poly(lactic-co-glycolic acid)
(PLGA), or a combination thereof.
13. The composition of claim 11, wherein the at least one
biodegradable porous particle facilitates or controls at least one
of intracellular delivery of an active agent, bio-distribution of
an active agent, stability of an active agent, and internalization
of the porous particle by one or more cells or organelles.
14. The composition of claim 1, wherein the at least one
biodegradable reinforcing particle comprises a degradation inducer
of one or more hydrogel porogen particles.
15. The composition of claim 14, wherein the one or more hydrogel
porogen particles comprise alginate.
16. The composition of claim 14, wherein the degradation inducer
comprises sodium citrate.
17. The composition of claim 1, wherein at least one biodegradable
reinforcing particle comprises a mesoporous silica particle.
18. The composition of claim 17, wherein the mesoporous silica
particle further comprises at least one active agent, at least one
imaging agent, or a combination thereof.
19. The composition of claim 18, wherein the at least one imaging
agent comprises barium sulfate.
20. The composition of claim 1, wherein at least one biodegradable
reinforcing particle comprises elongated, rod-like microparticles,
nanoparticles, or a combination thereof.
21. The composition of claim 1, wherein the at least one
biodegradable reinforcing particle is bound to the biodegradable
matrix.
22. The composition of claim 21, wherein the at least one
biodegradable reinforcing particle is covalently bound to the
biodegradable matrix through an acrylate moiety on a surface of the
at least one biodegradable reinforcing particle.
23. The composition of claim 22, wherein the biodegradable
reinforcing particle further comprises at least one active
agent.
24. The composition of claim 23, wherein at least one active agent
is selected from the group consisting of therapeutics, imaging
agents, anti-inflammatory agents, antibiotics, proteins,
platelet-rich plasma, cells, degradation inducers of porous
particles, and combinations thereof.
25. A composition comprising: (a) a biodegradable polymer matrix
comprising poly(propylene fumarate); (b) a population of
biodegradable reinforcing particles, dispersed within the
biodegradable polymer matrix, and comprised of porous oxide
particles, porous semiconductor particles, or a combination
thereof; (c) a population of hydrogel porogen particles dispersed
within the biodegradable matrix, and comprised of
poly(lactic-co-glycolic acid) (PLGA); and (d) at least one active
agent.
26. The composition of claim 25, wherein the at least one active
agent is comprised within one or more of the biodegradable
reinforcing particles, or within the population of hydrogel porogen
particles.
27. The composition of claim 25, wherein the population of hydrogel
porogen particles comprises at least one of alginates, fibrins, and
gelatins.
28. The composition of claim 25, wherein the at least one active
agent comprises an imaging agent, a therapeutic agent, or a
combination thereof.
29. The composition of claim 25, formulated as an injectable.
30. A pharmaceutical formulation, suitable for human injection,
that comprises: (a) a biodegradable polymer matrix comprising
poly(propylene fumarate); (b) at least one biodegradable
reinforcing particle dispersed in the polymer matrix, wherein the
at least one biodegradable reinforcing particle is selected from
the group consisting of porous oxide particles and porous
semiconductor particles; and (c) a population of hydrogel porogen
particles, dispersed within the biodegradable polymer matrix, that
comprise poly(lactic-co-glycolic acid) (PLGA), and at least one
therapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/270,324, filed Sep. 20, 2016, which is a continuation of
U.S. application Ser. No. 13/809,291, filed Mar. 22, 2013, which is
a National stage entry of PCT/US2011/029832 filed Mar. 24, 2011,
which in turn claims priority to U.S. Provisional Patent
Application No. 61/363,835, filed on Jul. 13, 2010 and U.S.
Provisional Patent Application No. 61/363,126, filed on Jul. 9,
2010. The entirety of each of the above-identified applications is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Current compositions and methods for tissue engineering or
wound healing through the use of scaffolds suffer from various
limitations. Such limitations may include insufficient
biocompatibility, insufficient biodegradability, lack of mechanical
stability, and insufficient porosity for the delivery of active
agents. Therefore, there is currently a need to develop new methods
and compositions for tissue engineering and wound healing that
address the aforementioned limitations.
BRIEF SUMMARY OF THE INVENTION
[0004] In some embodiments, the present invention provides
compositions that comprise: (1) a biodegradable polymer matrix
(e.g., an unsaturated biodegradable polymer, such as poly(propylene
fumarate) (PPF)); and (2) at least one biodegradable reinforcing
particle that is dispersed in the matrix. In some embodiments, the
biodegradable reinforcing particle is selected from the group
consisting of porous oxide particles and porous semiconductor
particles (e.g., mesoporous silica particles). In additional
embodiments, the compositions of the present invention further
comprise a (3) porogen particle that is also dispersed in the
matrix. In various embodiments, such porogen particles may be
hydrogels (e.g., alginates, fibrins, and gelatins), natural or
synthetic biodegradable particles, biodegradable porous particles
(e.g., silicon porous particles), and biocompatible vesicles (e.g.,
liposomes and/or micelles).
[0005] In further embodiments, the compositions of the present
invention are associated with one or more active agents. In various
embodiments, the active agents are associated with the
biodegradable polymer matrix, the biodegradable reinforcing
particle, and/or the porogen particle. In some embodiments, the
active agent comprises therapeutics, antibiotics, proteins,
platelet rich plasma (PRP), cells (e.g., stem cells), degradation
inducers of porogen particles (e.g., lactic acid and/or sodium
citrate), anti-inflammatory agents, cell viability enhancing agents
(e.g., glucose), and/or imaging agents (e.g., barium sulfate).
[0006] In various embodiments, the compositions of the present
invention may be utilized as scaffolds, such as scaffolds for
treating bone defects. Accordingly, in some embodiments, the
present invention also pertains to methods of treating a bone
defect in a subject by applying to an area of the bone defect in
the subject a scaffold of the present invention. Further
embodiments of the present invention pertain to methods of making
the compositions of the present invention.
[0007] The methods and compositions of the present invention have
numerous applications and advantages. For instance, in various
embodiments, the compositions and methods of the present invention
may be used in the treatment of bone defects, wound healing, tissue
engineering, and the prevention or treatment of microbial
infections.
BRIEF DESCRIPTION OF THE FIGURES
[0008] In order that the manner in which the above recited and
other advantages and objects of the invention are obtained, a more
particular description of the invention briefly described above
will be rendered by reference to specific embodiments thereof,
which are illustrated in the appended Figures. Understanding that
these Figures depict only typical embodiments of the invention and
are therefore not to be considered limiting of its scope, the
invention will be described with additional specificity and detail
through the use of the accompanying Figures in which:
[0009] FIGS. 1A-1F present images of alginate hydrogel
microspheres/beads (.about.200-500 microns). Inside the beads is
platelet-rich plasma (PRP) as well as mesenchymal stem cells (FIG.
1A-1B: optical image; FIG. 1C: confocal image green--cells; FIGS.
1D-1F are scanning electron microscopy (SEM) images).
[0010] FIG. 2 shows poly(lactic-co-glycolic acid) (PLGA) coated
mesoporous silicon.
[0011] FIGS. 3A-3D show images of surface-modified and
co-condensated silica nanorods SEM (FIGS. 3A-3B) at two different
magnifications and transmission electron microscope (TEM) (FIG.
3C-3D) at two different magnifications.
[0012] FIG. 4 illustrates the injectability of poly(propylene
fumarate) (PPF).
[0013] FIG. 5 is a photograph of an alginate/PPF composite scaffold
fabricated using a cylindrical Teflon mold.
[0014] FIGS. 6A-6F illustrate the operation of a biodegradable
scaffold to treat a bone defect.
[0015] FIG. 6A shows the integration of matrix components.
[0016] FIG. 6B shows the injection of the bioactive matrix into the
bone defect;
[0017] FIG. 6C shows the area of the bone defect 1 week after
injection. Degradation of alginate porogens and delivery of cells
and SEs to the surrounding scaffold can be seen.
[0018] FIG. 6D shows the area of the bone defect 2 weeks after
injection. Degradation of silicon enclosures (SEs) and
microparticles or nanoparticles (MSNs) and initial vascularization
can be seen.
[0019] FIG. 6E shows the area of the defect 3 weeks after
injection. Woven bone formation can be seen.
[0020] FIG. 6F shows a remodeled bone that is formed after the
completion of treatment.
[0021] FIGS. 7A-7F show various experimental results and schemes
related to calcium alginate bead production.
[0022] FIGS. 7A-7B show calcium alginate beads produced without PRP
(FIG. 7A), or with PRP (FIG. 7B).
[0023] FIGS. 7C-7E depict a scheme for production of calcium
alginate beads by an internal gelation/emulsion technique.
Insoluble calcium complex is dispersed in the aqueous phase
containing sodium alginate and bioactive components (FIG. 7C). The
aqueous phase is added to the oil phase with a surfactant present.
Continuous stirring forms a stable emulsion (FIG. 7D). An oil
soluble acid is then added to the mixture, thereby reducing the pH
and triggering the release of calcium ions from the calcium complex
to initiate gelation of the formed microspheres (FIG. 7E).
[0024] FIG. 7F shows the size distribution of the formed calcium
alginate beads. The peak size is in the range of 250 to 400
microns.
[0025] FIGS. 8A-8B show elastic modulus (FIG. 8A) and stress (FIG.
8B) at offset yield of composite putty containing 40% alginate
porogens and porous PPF scaffolds. The results are compared to
native human trabecular bone.
[0026] FIG. 9 presents a temperature profile of PPF cross-linking
with varying amounts of alginate porogens.
[0027] FIGS. 10A-10O show various studies relating to the
physiological effects of alginate porogens.
[0028] FIGS. 10A-10B show PDGF (platelet-derived growth factor),
VEGF (vascular endothelial growth factor) (FIG. 10C), and RANTES
(Regulated on Activation, Normal T Expressed and Secreted) (FIG.
10D), release from PRP within alginate porogens of varying
sizes.
[0029] FIG. 10E shows the effects of PRP released from PRP loaded
alginate porogens on cell proliferation over a three day
period.
[0030] FIG. 10F shows the effects of PRP release from PRP loaded
alginate porogens on cell migration. DAPI stained cells that
migrated through an 8 micron transwell towards the released
chemokines are shown on the top panel. Cell hemacytometer count of
cells that migrated through the 8 micron transwell over the period
of 4 days is illustrated in the graph on the bottom panel.
[0031] FIG. 10G shows subcutaneous implantation of calcium alginate
porogens clotted in a fibrin matrix in rats, vascularization of the
scaffold at 2 weeks (FIGS. 10H-I), H&E stain (FIG. 10J), and
Goldner Trichrome stain (FIG. 10K) of histological section from the
scaffold. The results indicate vessel formation and premature
collagen formation (green).
[0032] FIG. 10L shows viability staining of stem cells cryo-freezed
in alginate porogens after thawing. Live cells are shown in green,
and dead cells are shown in red. The bottom panel shows a trypan
blue exclusion count of viable cells after cryopreservation and
thawing (FIG. 10M).
[0033] FIG. 10N shows calcium alginate accelerated degradation
using sodium citrate as a chelation agent at various concentrations
(top panel). Cytotoxicity of Alginate/PRP porogens from the top
panel on rat cortical bone mesenchymal cells (MSC) are shown on the
bottom panel (FIG. 10O).
[0034] FIGS. 11A-11C demonstrate an increase in aspect ratio and a
decrease in the size of alginate porogen beads through the
adjustment of tetraethyl orthosilicate (TEOS) (FIG. 11A), cetyl
trimethyl ammonium bromide (CTAB) (FIG. 11B), and ammonia (FIG.
11C).
[0035] FIG. 12 presents results of measuring zeta potential for
co-condensated acrylate nanorods showing a surface charge of around
-17 mV. Oxidized silica (unmodified) has a surface charge of around
-40 mV.
[0036] FIG. 13 presents Fourier transform infrared spectrum (FTIR)
for post-modified acrylate nanorods showing C.dbd.O peaks at 1716
cm.sup.-1 and C.ident.C peaks at 1621 cm.sup.1.
[0037] FIGS. 14-16 show silica nanorods with 2.5% trimethoxysilyl
propyl methacrylate (FIG. 14); 5% trimethoxysilyl propyl
methacrylate (FIG. 15); and 10% trimethoxysilyl propyl methacrylate
(FIG. 16).
[0038] FIG. 17 presents results of measuring zeta potential for
co-condensated acrylate particles showing a surface charge of
around -17 mV. Oxidized silica (unmodified) has a surface charge of
around -40 mV.
[0039] FIG. 18 presents an FTIR for post modified acrylate nanorods
showing C.dbd.O peaks at 1716 cm.sup.-1 and C.ident.C peaks at 1621
cm.sup.1.
[0040] FIGS. 19A-19B present Brunauer-Emmett-Teller (BET) data
showing the pore size distribution of silica nanorods to be around
2.56 nm.
[0041] FIGS. 20A-20C demonstrate biocompatibility of mesoporous
silica.
[0042] FIG. 20A shows high cell viability after 24 hours of
treatment with silica concentrations of about 0.01% by weight. The
silica used in this experiment are washed E (post modified) and
washed CC (co-condensated).
[0043] FIG. 20B shows viable cell count of MDA231 cells incubated
with mesoporous nanorods (MSNRs). The cells are stained with
Annexin V, which is indicative of apoptosis.
[0044] FIG. 20C shows MTT assays of human umbilical vein
endothelial cells (HUVEC) incubated with MSNRs.
[0045] FIG. 21 shows an X-ray image of agarose composites with
different concentrations of silica nanorods containing barium
sulfate (from right to left: 0%, 0.5% and 2%).
[0046] FIG. 22 shows GIF images through TEM showing the presence of
barium and sulfur within silica nanorods.
[0047] FIG. 23 is a graph demonstrating controlled release of a
model drug, DOX-HCl, from silica nanorods.
[0048] FIG. 24 is a graph demonstrating controlled release of
Cefazolin from silica nanorods.
[0049] FIGS. 25A-25B show various data related to the mechanical
strength of silica nanorods.
[0050] FIG. 25A shows the stress offset of control, 2.5%
co-condensated silica nanorods (CC) and post-modified silica
(E)
[0051] FIG. 25B shows the compressive modulus of 2.5%
co-condensated silica nanorods (CC) and post-modified silica when
dispersed in PPF polymer.
[0052] FIGS. 26A-26C show data relating to the mineralization of
various scaffolds, and the use of PPF in various compositions.
[0053] FIG. 26A shows data relating to the mineralization of
agarose-coated silica nanoparticle scaffolds.
[0054] FIG. 26B shows images from a mineralization study with Rat
Compact Bone stromal cells after 3 weeks.
[0055] From left to right the first panel_shows a phase image of
matrigel alone in osteogenic media;
[0056] the second panel shows a phase image of matrigel and mSNR in
osteogenic media; and
[0057] the third panel shows a phase image of matrigel and mSNR, in
osteogenic media. The sample was stained with Von Kossa stain for
calcium-phosphate mineral (brown) and alkaline phosphatase enzyme
activity (blue). The sample was also stained with the nuclear
counterstain, nuclear fast red (pink).
[0058] FIG. 26C shows temperature increase due to PPF injectable
putty cross-linking.
[0059] FIG. 27 shows cefazolin release from gelatin-coated
mesoporous silicon (MPS).
[0060] FIG. 28 shows cefazolin release from agarose coated MPS.
[0061] FIG. 29 shows cefazolin release from APTES coated MPS.
[0062] FIGS. 30A-30C show results from the flow cytometry analysis
of MPS (FIG. 30A: 2000.times.g rcf; FIG. 30B: 10000.times.g rcf;
FIG. 30C: 26000.times.g rcf). Data are from one experiment
representative of three. SSC: side scatter; FSC: forward
scatter.
[0063] FIG. 31 shows results from multisizer analysis of the
MPS.
[0064] FIG. 32 presents results of FACS analysis of geometric mean
X value of MPS Size Distribution.
[0065] FIG. 33 presents results of FACS analysis of geometric mean
Y value of MPS Size Distribution.
[0066] FIGS. 34A-34B presents zeta potential of different surface
modified MPS.
[0067] FIGS. 35A-35J show agarose modification of nanoporous
silicon particles (NSP): NSP observed with SEM at low (FIGS.
35A-35E) and high (FIGS. 35F-35J) magnification: (a and A) bare NSP
and (b, c, d and e) agarose coated NSP with different agarose
concentration (0.05, 0.125, 0.25 and 0.5% respectively).
[0068] FIGS. 36A-36H show silicon particles (NSP) degradation: SEM
observation at different times of NSP: bare NSP after (FIG. 36A) 2
hours, (FIG. 36B) 4 hours, (FIG. 36C) 8 hours, (FIG. 36D) 12 hours,
(FIG. 36E) 1 day, (FIG. 36F) 2 days, (FIG. 36G) 3 days, and (FIG.
36H) 4 days of incubation with PBS. Scale bar is 1 m.
[0069] FIGS. 37A-37P show particles degradation as measured through
FACS: (FIGS. 37A, 37D, 37H, and 37K) Forward and side scattering
data analysis for bare (NC) compared with nanoporous silicon
particles coated two agarose concentrations (0.05% agarose
concentration (A1) (FIG. 37B, FIG. 37E, FIG. 371, and FIG. 37L) and
0.125% agarose concentration (A2) (FIG. 37C, FIG. 37F, FIG. 37J,
and FIG. 37M), over time (1 h-72 h); (FIG. 370) size measurements
over time of bare (NC) and agarose coated nanoporous silicon
particles with two agarose concentrations (0.05% and 0.125%, A1 and
A2 respectively); and (FIG. 37P) shows the correlation.
[0070] FIGS. 38A-38C show protein load and release: (FIG. 38A)
amount of BSA loaded in bare (NC) and agarose coated particles with
two agarose concentrations (0.125 and 0.05%, A1 and A2,
respectively); (FIG. 38B) fluorescence of agarose coated (A1 and
A2) and NC nanoporous silicon particles (NSP), as measured by FACS,
and (FIG. 38C) BSA released from agarose coated (A1 and A2) and NC
NPS, as measured with spectrofluorimetry.
[0071] FIGS. 39A-39D show gel electrophoresis: (FIG. 39A) SDS-page
of protein solution released after 24 hours from bare (NC) and
agarose coated (Ag) nanoporous silicon particles treated for
different times with trypsin (treatment duration in minutes,
printed in white on each column). 1, 2 and 3 indicate the most
abundant digestion products. (FIG. 39B, FIG. 39C, FIG. 39D)
SDS-page relative intensity quantification with ImageJ of trypsin
(Tryp), BSA and the three most abundant digestion products (Dig.1,
2 and 3) detected in the protein solution released after 24 hours
from bare (NC) and agarose coated (Ag--composition 0.125%) silicon
particles after different trypsin treatment duration.
[0072] FIGS. 40A-40B show released protein solution chromatography
through high pressure liquid chromatography (HPLC) analysis of BSA
solution released after 24 hours by (FIG. 40A) NC and (FIG. 40B) Ag
particles not treated (blue-1) and treated with trypsin for 15
minutes, 2 hours, 4 hours, 8 hours and 18 hours (green-2, light
blue-3, brown-4, light green-5 and pink-6, respectively). Arrows
point to three digestion products, the amount of which increases
with trypsin treatment time.
[0073] FIGS. 41A-41F show in vitro confocal study of cellular
internalization of silicon particles (NSP) and protein uptake:
(FIG. 41A--control) cells (HUVEC) incubated for 48 hours without
NSP; (FIG. 41B) with BSA loaded NSP added into the media; (FIG.
41C) with agarose coated BSA loaded NSEs added into the media;
(FIG. 41D) with NC and (FIG. 41E) Ag BSA loaded NSP placed in a
transwell on top of the cells, or (FIG. 41F) in BSA solution. White
scale bar is 50 .mu.m in FIG. 41A-41C and 10 .mu.m in FIG.
41D-41F.
[0074] FIG. 42 shows pH measurement of acid solution change due to
agarose coating solution.
[0075] FIGS. 43A-43F show confocal study of cellular uptake of
protein from internalized particles (NSP): HUVEC incubated for
(FIG. 43A, FIG. 43C, respectively) 24 hours and (FIG. 43B, FIG. 43D
respectively) 48 hours with (FIG. 43A, FIG. 43B) FITC-BSA loaded
agarose coated NSP, (43C, 43D) FITC-BSA loaded not coated NSP.
Scale bar is 10 .mu.m. FIG. 43E and FIG. 43F show quantification of
uptake of BSA within the cells: fluorescence intensity within (FIG.
43E) the nucleus and (FIG. 43B) the cytoplasm of the cells
quantified with NIS-Elements. Red square and blue triangle refer to
NC and Ag NSP, respectively.
[0076] FIGS. 44A-44D are schematic diagrams of PLGA/pSi
microspheres fabrication through the S/O/W emulsion method. (FIG.
44A) PLGA/pSi suspension was poured into water phase. (FIG. 44B)
The suspension was emulsified in the water phase. (FIG. 44C)
Surfactants were added to stabilize the structures. (FIG. 44D)
Cartoon depicting the final composition of a PLGA/pSi microsphere
(components not in scale).
[0077] FIGS. 45A-45D show an SEM image of pSi particles at: (FIG.
45A) lower magnification showing particle uniformity in size and
shape; and (FIG. 45B) a higher magnification micrograph revealing
the pore structure as seen on the surface of the particle. Low
power micrographs illustrate: (FIG. 45C) the front; and (FIG. 45D)
rear surfaces of a pSi particle.
[0078] FIGS. 46A-46D show a physical characterization and size
distribution of PLGA/pSi microspheres. (FIG. 46A) SEM image of
presorted microspheres. (FIG. 46B) An optical microscopy image
shows the presence of pSi particles (arrows) enclosed in the larger
PLGA spheres. (FIG. 46C) Fluorescence microscope, and (FIG. 46D)
the size distribution of PLGA/pSi microspheres displays the uniform
product centered around 24.5 .mu.m.
[0079] FIGS. 47A-47E show a FACS analysis of nonsorted and sorted
PLGA/pSi microspheres prepared with 488-DyLight conjugated pSi
particles (DyLight-PLGA/pSi microspheres): (FIG. 47A) the percent
of DyLight-PLGA/pSi microspheres in the non-sorted, sorted
microspheres and supernatant; (FIG. 47B) the mean fluorescence of
the sorted, nonsorted microspheres, and the supernatant; (FIG. 47C)
fluorescence intensity and distribution of 488-DyLight conjugated
pSi particles (light green), nonsorted microspheres (blue), sorted
microspheres (dark green), and supernatant solution (black). Also
shown are confocal images of (FIG. 47D) nonsorted microspheres and
(FIG. 47E) sorted microspheres.
[0080] FIGS. 48A-48D show release profiles of FITC-BSA from various
examined PLGA/pSi microsphere formulations, including: (FIG. 48A)
total FITC-BSA released over 27 days; (FIG. 48B) first three day
release; (FIG. 48C) day 5 to 15 release; and (FIG. 48D) day 15 to
27 release.
[0081] FIGS. 49A-49B show PLGA and PLGA/pSi microspheres analyzed
via FACS during in vitro release.
[0082] FIG. 49A shows histographic overlay of the fluorescence
intensity and distribution of control PLGA (left) and PLGA/pSi
(right) over 2 weeks of incubation in PBS.
[0083] FIG. 49B shows decrease of fluorescence intensity as
measured through FACS dropped to minimum at day 3 in control PLGA.
PLGA/pSi showed slow decrease in fluorescence intensity and
displayed 3 fold the intensity of control at 2 weeks.
[0084] FIGS. 50A-50H and FIGS. 50J-50P show SEM images of PLGA/pSi
microsphere degradation over 1, 2, 3, 4, and 6 weeks with 6%
coating (FIGS. 50A-50E), 10% coating (FIGS. 50F-50H and FIGS.
50J-50K), or 20% coating (FIGS. 50L-50P).
[0085] FIGS. 51A-51B show the pH of pSi, PLGA, and PLGA/pSi
microsphere degradation byproducts in PBS at 37.degree. C. over 4
weeks for (FIG. 51A) PLGA-only microspheres (control) and (FIG.
51B) PLGA/pSi microspheres.
[0086] FIG. 52 shows FITC-BSA degradation over 2 weeks. SPS-PAGE of
release products showed BSA (approximately 68 kDa) released from
PLGA/pSi microspheres suffered no degradation bands compared to
controls (BSA in solution for 7 and 14 days, columns 2 and 3,
respectively).
[0087] FIGS. 53A-53G show mineralization on the surface of PLGA/pSi
microspheres, including SEM images of (FIG. 53A, FIG. 53C) PLGA and
(FIG. 53B, FIG. 53D) PLGA/pSi microspheres in osteogenic media
after 3 and 21 days, respectively. Also shown are (FIG. 53E-FIG.
53F) SEM images at day 21 at higher magnification. In addition, the
(FIG. 53G) EDX spectrum of mineralized PLGA/pSi microspheres on day
3 (gray dot line) and day 14 (black solid line) is shown.
[0088] FIGS. 54A-54J show confocal microscopy images of PLGA/pSi
microparticles (loaded with green fluorescent BSA) were not
internalized by bone marrow derived stromal cells (BMSCs) at (FIGS.
54D-54G) 0 hour, (FIGS. 54E-54H) 48 hours, or (FIGS. 54F-54I) 120
hours, while (FIG. 54G) pSi microparticles were internalized by
BMSCs at 0.5 hours (FIG. 54A), 48 hours (FIG. 54B), and 120 hours
(FIG. 54C).
[0089] FIG. 54J shows a schematic diagram of the mechanism of
action of PLGA/pSi microspheres compared to pSi. Following
internalization, pSi is trapped within lysosomes, while the
PLGA/pSi particles are not endocytosed by BMSC and release their
payload outside the cells where it can exert its bioactive function
and trigger nuclear changes through the classic mechanism of signal
cascade.
[0090] FIGS. 55A-55E show confocal images of stained HUVEC
(green-BSA, red-actine filaments, blue-nuclei) after 7 days in
culture (FIG. 55A, FIG. 55B--control, BSA in solution), or after
incubation with BSA loaded PLGA/pSi microspheres (FIG. 55C, FIG.
55D), with overlap of all three fluorescent channels (FIG. 55A and
FIG. 55C), or bright field and green channels (FIG. 55B and FIG.
55D). Average fluorescence intensity of the three fluorescent
channels (FIG. 55E) related to control HUVEC (dark color bars) and
HUVEC incubated with PLGA/pSi (light color bars) as measured at the
confocal microscope are also shown.
[0091] FIG. 56 shows an analysis of the porous structure of MP2
porous silicon microparticles by nitrogen adsorption-desorption
isotherms at 77K. The inset graph shows pore size distribution
according to the Barrett-Joyner-Halenda model.
[0092] FIG. 57 shows Zeta potential analysis indicating that the
oxidized pSi surface had a surface charge of -30.39 My (left
panel), while the APTES modified pSi particles had a value of 6.44
mV (right panel).
[0093] FIG. 58 shows the loading efficiency of seven different
types of pSi particles. For a certain concentration of FITC-BSA
solution, mesoporous silicon particles has the highest loading
efficiency.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0094] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only, and are not restrictive of the invention, as
claimed. In this application, the use of the singular includes the
plural, the word "a" or "an" means "at least one", and the use of
"or" means "and/or", unless specifically stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements or
components comprising one unit and elements or components that
comprise more than one unit unless specifically stated
otherwise.
[0095] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated herein by reference in their entirety for any purpose.
In the event that one or more of the incorporated literature and
similar materials defines a term in a manner that contradicts the
definition of that term in this application, this application
controls.
Definitions
[0096] Unless otherwise specified "a" or "an" means one or
more.
[0097] "Microparticle" means a particle having a maximum
characteristic size from 1 micron to 1000 microns, or from 1 micron
to 100 microns.
[0098] "Nanoparticle" means a particle having a maximum
characteristic size of less than 1 micron.
[0099] "Nanoporous" or "nanopores" refers to pores with an average
size of less than 1 micron.
[0100] "Biodegradable material" refers to a material that can
dissolve or degrade in a physiological medium, such as PBS or
serum.
[0101] "Biocompatible" refers to a material that, when exposed to
living cells, will support an appropriate cellular activity of the
cells without causing an undesirable effect in the cells such as a
change in a living cycle of the cells; a release of proinflammatory
factors; a change in a proliferation rate of the cells and a
cytotoxic effect.
[0102] APTES stands for 3-aminopropyltriethoxysilane.
[0103] Loading capacity or loading efficiency refers to an amount
of a load that can be contained in pores of a porous object.
[0104] Introduction
[0105] Insufficient healing occurring in cases of traumatic
fractures or injuries may be substantial. For instance, severe leg
injuries are typically repaired with bone grafts. Pins, plates or
screws hold the grafts to healthy bone while external fixation
provides support. However, it may take months to years before the
injured patient fully recovers. Therefore, a technology that
provides both immediate mechanical stability to restore function
and accelerates the regeneration process is needed.
[0106] The ideal tissue engineering scaffold may require several
characteristics. Such characteristics may include biocompatibility,
biodegradability, mechanical stability, interconnected porosity and
the ability to deliver active agents, such as therapeutic and/or
imaging agents. To achieve such properties, one may combine diverse
technologies into a multi-functional composite materials. For
instance, it is well known in the field of tissue engineering (TE)
that the porosity and pore interconnectivity of the scaffold may be
essential for tissue in-growth, vascularization and nutrient
supply. However, high porosity may severely compromise mechanical
properties. The challenge may lie in the trade-off between porosity
and mechanical integrity, wherein porosity is usually negatively
correlated with mechanical strength.
[0107] The present disclosure presents a strategy for conquering
the challenge of meeting mechanical requirements of tissue
engineering scaffolds while maintaining the porous structure
necessary for tissue integration and supplying of essential
bioactive molecules for accelerated tissue regeneration. In some
embodiments, the present invention provides compositions that
comprise: (1) a biodegradable polymer matrix; and (2) at least one
biodegradable reinforcing particle that is dispersed in the matrix.
In additional embodiments, the compositions of the present
invention further comprise a (3) porogen particle that is also
dispersed in the matrix. In further embodiments, one or more of the
above-mentioned individual components are associated with an active
agent.
[0108] In further embodiments, the compositions of the present
invention may be utilized as scaffolds, such as scaffolds for
treating bone defects. Accordingly, in various embodiments, the
present invention also provides methods of treating a bone defect
in a subject by applying to an area of the bone defect in the
subject a scaffold of the present invention. Further embodiments of
the present invention pertain to methods of making the compositions
of the present invention.
[0109] As discussed in more detail below, the methods and
compositions of the present invention have numerous applications
and advantages. More detailed aspects of various embodiments of the
present invention will now be described below as specific and
non-limiting examples.
[0110] Biodegradable Polymer Matrix
[0111] In the present invention, biodegradable polymer matrices
generally refer to polymer-based matrices that show at least some
biodegradability. In various embodiments, the biodegradable polymer
matrices of the present invention may comprise a biodegradable
polymer. Non-limiting examples of suitable biodegradable polymers
include collagen, gelatin, alginate, polycaprolactone, and
poly(lactic-co-glycolic acid) (PLGA).
[0112] In many embodiments, the biodegradable polymer may be an
unsaturated biodegradable polymer (i.e. a biodegradable polymer
containing at least one unsaturated carbon-carbon bond, such as a
double or a triple bond). Such unsaturated polymers may be
cross-linkable in situ. Non-limiting examples of unsaturated
biodegradable polymers include poly(propylene fumarate) (PPF),
poly(E-caprolactone-fumarate), and mixtures and co-polymers
thereof.
[0113] Additional biodegradable polymers that may be used in the
polymer matrices of the present invention are disclosed, for
example, in WO 2010/040188; WO2006/023130; WO1997/045532;
US2005/0177249; US2006/026335; U.S. Pat. Nos. 6,858,229, 5,522,895,
6,281,257, and 6,124,373; Mano et al. Composites Science and
Technology. 2004. 64:789-817; Rezwan et al. Biomaterials. 2006.
27:3413; Boccaccini et al. Expert Review of Medical Devices. 2005.
2(3):303; Advanced Drug Delivery Reviews. 2007. 59 (4-5):249; and
Tan et al. Materials. 2010. 3:1746; J R Soc. Interface. 2007.
4(17): 999-1030.
[0114] As set forth in more detail below, the biodegradable polymer
matrices of the present invention may be associated with one or
more active agents. Furthermore, the biodegradable polymer matrices
of the present invention may be associated with biodegradable
reinforcing particles and porogen particles.
[0115] In a more specific embodiment, the biodegradable polymer
matrix comprises PPF. By way of background PPF is an example of an
in situ cross-linkable polymer that exhibits mechanical properties
close to the ones of the trabecular bone. See, e.g. Peter et al. J
Biomed Mater Res. 1999; 44: 314-21. As set forth in more detail
below, these properties of PPF's in the biodegradable polymer
matrices of the present invention may be further amplified by the
use of biodegradable reinforcing particles, porogen particles and
active agents.
[0116] Biodegradable Reinforcing Particles
[0117] In the present invention, biodegradable reinforcing
particles generally refer to particles of the nano scale that
provide mechanical strength to the surrounding polymer matrix.
Biodegradable reinforcing particles may also simultaneously release
active agents upon biodegradation. Generally, the biodegradable
reinforcing particles of the present invention are dispersed in the
biodegradable polymer matrix and selected from the group consisting
of porous oxide particles and porous semiconductor particles.
[0118] A person of ordinary skill in the art will recognize that
various suitable biodegradable reinforcing particles may be used in
the present invention. Non-limiting examples of suitable
biodegradable reinforcing particles include biodegradable oxide
microparticles or nanoparticles (e.g., silica particles), or
biodegradable semiconductor microparticles or nanoparticle (e.g.,
silicon particles). In many embodiments, the biodegradable
reinforcing particles of the present invention may comprise porous
or mesoporous microparticles or nanoparticles, such as mesoporous
silica or silicon particles. In some embodiments, the biodegradable
reinforcing particles may comprise nanoporous microparticles or
nanoparticles.
[0119] In various embodiments, the biodegradable reinforcing
particles of the present invention may also be associated with an
active agent. Non-limiting examples of suitable active agents
include degradation inducers of the porogen particles, imaging
agents (e.g., barium sulfate), proteins, platelet rich plasma, cell
viability enhancing agents (e.g., glucose), anti-inflammatory
agents, antibiotics, therapeutic agents, growth factors, DNA,
siRNA, and the like. Such active agents may also include: agents
that can prevent an infection at a site of a bone defect, such as a
bone fracture; agents that can contribute to bone regeneration at a
site of a bone defect; and agents that can contribute to cell
viability at a site of a bone defect.
[0120] In more specific embodiments, the biodegradable reinforcing
particles of the present invention contain an imaging agent, which
may facilitate imaging and/or monitoring of a site of a bone
defect, such as a bone fracture. One non-limiting example of such
an imaging agent may be barium sulfate, which may facilitate X-ray
imaging of a bone defect site.
[0121] In more specific embodiments, biodegradable reinforcing
particles that comprise porous microparticles or nanoparticles
(reinforcing microparticles or nanoparticles) may contain one or
more active agents, such as therapeutic and/or imaging agents that
may be released upon the degradation of the particles. The active
agents which may be contained inside the reinforcing microparticles
or nanoparticles may include, without limitation, antibiotics,
anti-inflammatory agents, proteins (such as growth factor), and
nucleic acids (such as DNA and siRNA). In some embodiments, the
growth factors may include one or more of PDGF.alpha..beta.,
PDGF.alpha..alpha., PDGF.beta..beta., TGF1, TGF2, vascular
endothelial growth factor (VEGF) and/or epithelial growth factor
(EGF).
[0122] In some embodiments, the reinforcing microparticles or
nanoparticles may contain one or more proteins, such as fibrin,
fibronectin and vitronectin. In some embodiments, the reinforcing
microparticle or nanoparticle may contain platelet-rich plasma
(PRP). PRP may contain PDGF.alpha..beta., PDGF.alpha..alpha.,
PDGF.beta..beta., TGF1, TGF2, VEGF, EGF, fibrin, fibronectin and/or
vitronectin. In some embodiments, the reinforcing microparticles or
nanoparticles may contain a cell viability enhancing agent, which
may be, for example, a sugar, such as glucose or glucose
derivative, such as glucose lactam. In some embodiments, the
reinforcing microparticles or nanoparticles may contain a bone loss
preventing agent, which may be, for example, a biphosphonate, such
as etidronate, clodronate, tiludronate, pamidronate, neridronate,
olpadronate, alendronate, ibandronate, risendronate, or
zolendronate.
[0123] In some embodiments, the reinforcing microparticles or
nanoparticles may contain one or more imaging agents, which may be
used for imaging or monitoring the treated bone defect site. Such
imaging agents may include, but not be limited to X-ray contrast
agents, such as barium sulfate; MRI contrast agents; ultrasound
contrast agents; fluorescent agents, such as fluorescent dyes and
quantum dots; and metal nanoparticles. In more specific
embodiments, the reinforcing microparticle or nanoparticle is a
mesoporous silica particle, and the active agent is an imaging
agent that is embedded into the matrix of the silica particle.
[0124] In various embodiments, the biodegradable reinforcing
particles may constitute from 1% to 30%, from 2% to 25%, from 3% to
20%, or from 5% to 15% of the volume of the composition (or any
sub-range within these ranges).
[0125] In many embodiments, biodegradable reinforcing particles may
be anisotropic particles (i.e. particles that have one of their
dimensions (e.g., length or thickness) substantially different from
the other two, which may define a cross-section of the
particle).
[0126] An aspect ratio of the biodegradable reinforcing particle
may be defined as a ratio between the length or thickness and its
mean lateral dimension (i.e., mean dimension of its cross-section).
Such a mean lateral dimension may be a diameter of a circular
cross-section, a side-length for a square cross-section, or a
square root of a product of two lateral dimensions for structures
that have a cross-section characterized by two dimensions (such as
a rectangular cross-section or an elliptical cross-section). For
the anisotropic particle, the aspect ratio is substantially
different than 1.
[0127] In many embodiments, the biodegradable reinforcing particle
may be an elongated, rod-like particle. In some embodiments, the
biodegradable reinforcing particles have an aspect ratio of at
least 2, at least 4, at least 10, at least 20, at least 50, at
least 100, at least 200, at least 500, or at least 1000. Such
elongated particles may be prepared using the methods disclosed in
the Examples below. Such elongated particles may be also be
prepared using the techniques disclosed in U.S. patent application
Ser. No. 13/044,250 and PCT Application No. PCT/US 11/27746.
[0128] In various embodiments, biodegradable reinforcing particles
of the present invention may have a cross-section having the
greater of lateral dimension(s) of no more than 10 microns, no more
than 5 microns, no more than 2 microns, no more than 1 micron, no
more than 500 nm, no more than 200 nm, no more than 100 nm, or no
more than 50 nm. In many embodiments, the smaller of the lateral
dimension(s) of the biodegradable reinforcing particles'
cross-section is no more than 5 microns, no more than 2 microns, no
more than 1 micron, no more than 500 nm, no more than 200 nm, no
more than 100 nm, or no more than 50 nm. In many embodiments, the
smaller of the lateral dimension(s) of the cross-section of the
biodegradable reinforcing particles may be no more than 5 microns,
no more than 2 microns, no more than 1 micron, no more than 500 nm,
no more than 200 nm, no more 100 nm, or no more than 50 nm.
[0129] The cross-section of the biodegradable reinforcing particles
of the present invention may have a variety of shapes. In some
embodiments, the cross-section may be circular or elliptical. In
some embodiments, the cross-section may be rectangular.
Considerations for selecting shapes and sizes of reinforcing
particles are disclosed, for example, in Ranganathan et al. Acta
Biometer. 2010. 6(9):3448-56. Epub 2010 Mar. 24.
[0130] In many embodiment, the biodegradable reinforcing particles
may be integrated in the biodegradable polymer matrix. Such
integration may be involve covalently binding the reinforcing
particles with the polymer matrix. For such covalent binding, the
reinforcing particles may comprise a chemical moiety that is
capable of covalently bonding to the biodegradable polymer. One
non-limiting example of such a chemical moiety may be an acrylate.
In some embodiments, the chemical moiety may be introduced on a
surface of the biodegradable reinforcing particles after the
particles are fabricated or synthesized. Yet, in some other
embodiments, the chemical moiety may be introduced into the
biodegradable reinforcing particles during their fabrication or
synthesis.
[0131] Porogen Particles
[0132] In the present invention, porogen particles generally refer
to biodegradable particles of the micron scale that are dispersed
in the polymer matrix. In addition, the porogen particles of the
present invention may be associated with one or more active agents.
Upon degradation, the porogen particles of the present invention
may leave interconnected porosity throughout the matrix while
simultaneously releasing active agents (e.g., cells). In addition,
the porogen particles of the present invention may contain within
them additional particles, such as biodegradable porous particles
(e.g., silicon porous particles).
[0133] In various embodiments, the porogen particles of the present
invention (including any particles within the porogen particles)
may be hydrogel porogen particles (e.g., alginates, fibrins, and
gelatins), natural or synthetic biodegradable particles (e.g.,
particles derived from or coated with poly(lactic-co-glycolic acid)
(PLGA)), biodegradable porous particles (e.g., silicon porous
particles), and biocompatible vesicles (e.g., liposomes and/or
micelles).
[0134] In various embodiments, a surface of a porogen particle (or
a surface of a particle within the porogen particle) is modified
with a biodegradable polymer. In some embodiments, the
biodegradable polymer is agarose. In further embodiments, the
biodegradable polymer is PLGA. In more specific embodiments,
porogen particles contain biodegradable porous particles within
them (e.g., silicon porous particles) that are coated with a
biodegradable polymer (e.g., PLGA and/or agarose).
[0135] Without being bound by theory, Applicants envision that
porogen particles of the present invention can help facilitate or
control various properties of the compositions of the present
invention. For instance, in some embodiments, porogen particles may
help facilitate or control the bio-distribution of active agents to
various parts of an organism (e.g., cells, tissues, organs,
etc.).
[0136] In further embodiments, porogen particles can help
facilitate or control the intracellular delivery of active agents
to various organelles (e.g., lysosomes, cytoplasm and nuclei). For
instance, in some embodiments, porogen particles can prevent or
guide particles to lysosomes, cytoplasms, nuclei or other cellular
organelles.
[0137] In addition, porogen particles may help facilitate the
internalization of the particle by various cells and organelles.
For instance, in some embodiments, an agarose coating on a
biodegradable porous particle within a porogen particle may help
enhance active agent delivery to the nuclei of cells.
[0138] In additional embodiments, porogen particles of the present
invention may help facilitate, preserve or control the stability of
active agents. For instance, in cases where the active agent is a
protein, porogen particles of the present invention may help
prolong protein stability by preserving protein structure over time
and protecting the protein from enzymatic digestion.
[0139] In some embodiments, the porosity and pore interconnectivity
within the compositions of the present invention may be created in
vivo using porogen particles. In some embodiments, the porogen
particles may have the ability to encapsulate cells, such as stem
cells, an/or active agents, such as therapeutic and/or imaging
agents. In some embodiments, the porogen particles may contain or
encapsulate one or more proteins. For example, in certain
embodiments, plateletrich plasma (PRP), which is a blood derived
liquid that may provide proper ECM-like protein for cellular
attachment and release one or more native factors from platelets to
recruit and proliferate cells, may be integrated or incorporated
within the porogen particles. Such incorporation may contribute to
obtaining a desired size of the porogen particles, which may be
from 100 microns to 700 microns, from 150 microns to 600 microns,
or from 200 microns to 500 microns. Such incorporation may also
provide a sustained release of growth factors from the porogen
particles.
[0140] In various embodiments, the porogen particles of the present
invention may degrade in a body of a patient with a rate faster
than a biodegradation rate of the biodegradable polymer of the
matrix, thereby forming a porous network in the matrix. In some
embodiments, such a porous network may be necessary for formation
of new vasculature at a bone defect site. In some embodiments, the
biodegradation rate of the porogen particles may be no more than 3
months, no more than 2 months, no more than 1 month, no more than 2
weeks, no more than 10 days, no more than 7 days, no more than 6
days, no more than 5 days, no more than 4 days, no more than 3
days, no more than 2 days, or no more than 1 day.
[0141] In various embodiments, the porogen particles of the present
invention may have a characteristic size, such as a diameter that
may range from 100 microns to 700 microns, from 150 microns to 600
microns, or from 200 microns to 500 microns. In further
embodiments, the porogen particles of the present invention may
constitute from 20% to 95%, from 25% to 90%, from 30% to 80%, or
from 35% to 75% of the volume of the composition.
[0142] In more specific embodiments, the porogen particles of the
present invention (or any particles within them) may be hydrogel
porogen particles (i.e. particles comprising a natural or synthetic
hydrogel). Examples of natural hydrogels include hydrogels based on
natural biodegradable polymers, such as gelatin or collagen.
Examples of synthetic hydrogels include hydrogels based on
synthetic biodegradable polymers, such as oligo(poly (ethylene
glycol) fumarate (OPF).
[0143] In some embodiments, the hydrogel material in the porogen
particle may comprise a polysaccharide polymer. In some
embodiments, the hydrogel material may be an anionic
polysaccharide, such as alginate. Various suitable porogen
particles (including alginate) are disclosed in WO2005/020849,
US2008/0206308, U.S. Pat. No. 6,656,508, US 2002/0001619, US
2002/0168406, WO2008/006658, WO2008/073856, EP1664168,
US2007/0178159, and WO2007/089997.
[0144] In some embodiments, the hydrogel-based porogen particles
may contain a metal ion, which may be replaced or dissociated from
the complex to facilitate a degradation of the porogen particle.
Such a replacement may be initiated by a degradation initiator,
such as a chelation agent. The degradation initiator may be
contained in one or more microparticles or nanoparticles, which may
be dispersed in the polymer matrix or contained inside the porogen
particle. In some cases, the microparticle or nanoparticle
containing the degradation initiator may be a reinforcement
microparticle or nanoparticle, as discussed above. In more specific
embodiments where the porogen is an alginate, the replaceable metal
ion in the hydrogel may be calcium, and the degradation initiator
may be a calcium chelating compound, such as a phosphate (e.g.,
sodium phosphate), a citrate (e.g., sodium citrate) or a lactate
(e.g., sodium lactate).
[0145] In addition to their porogenic role, the porogen particles
of the present invention may also serve as a delivery vehicle for
delivering one or more active agents, such as an imaging agent
and/or a therapeutic agent (as discussed above). In such a case,
one or more active agents may be contained inside the porogen
particle. In some embodiments, active agents may be contained in
particles within porogen particles.
[0146] Non-limiting examples of active agents which may be
contained inside the porogen particle may include antibiotics,
proteins (e.g., growth factors), platelet rich plasma, cells (e.g.,
stem cells), degradation inducers of porogen particles (e.g.,
lactic acid), anti-inflammatory agents, and nucleic acids (e.g.,
DNA and/or siRNA). In further embodiments, the porogen particle
associated with the active agent is a biodegradable porous
particle. In more specific embodiments, the porogen particles
comprise alginate, and the active agent is a degradation inducer
that comprises sodium citrate. In more specific embodiments, the
alginate-based particle containing sodium citrate is within another
porogen particle.
[0147] In further embodiments, the porogen particles may contain
one or more growth factors, such as PDGF.alpha..beta.,
PDGF.alpha..alpha., PDGF.beta..beta., TGF1, TGF2, vascular
endothelial growth factor (VEGF) and epithelial growth factor
(EGF). In some embodiments, the porogen particle may contain one or
more proteins, such as fibrin, fibronectin and vitronectin. Such
proteins may act as cell adhesion molecules for osteoconduction and
as matrix for bone, connective tissue and/or epithelial growth.
[0148] In some embodiments, the porogen particles may contain
platelet-rich plasma (PRP), which may be released from the porogen
matrix. PRP may contain PDGF.alpha..beta., PDGF.alpha..alpha.,
PDGF.beta..beta., TGF1, TGF2, VEGF, EGF, fibrin, fibronectin and/or
vitronectin.
[0149] In some embodiments, the porogen particles may contain
smaller size microparticles or nanoparticles, which may also
contain one or more active agents, such as those mentioned above.
Such microparticles or nanoparticles may be porous or mesoporous
microparticles or nanoparticles. Such porous or mesoporous
particles may be silicon or silica porous particles such as those
disclosed in one of the following documents: (1) PCT publication
no. WO 2007/120248 (published on Oct. 25, 2007); (2) PCT
publication no. WO 2008/041970 (published on Apr. 10, 2008); (3)
PCT publication no. WO 2008/021908 (published on Feb. 21, 2008);
(4) U.S. Patent Application Publication No. 2008/0102030 (published
on May 1, 2008); (5) U.S. Patent Application Publication No.
2003/0114366 (published on Jun. 19, 2003); (6) U.S. Patent
Application Publication no. 2008/0206344 (published on Aug. 28,
2008); (7) U.S. Patent Application Publication no. 2008/0280140
(published on Nov. 13, 2008); (8) PCT Patent Application
PCT/US2008/014001, filed on Dec. 23, 2008; (9) U.S. Pat. No.
6,107,102 (issued on Aug. 22, 2000); (10) U.S. Patent Application
Publication No. 2008/0311182 (published on Dec. 18, 2008); (11) PCT
Patent Application No. PCT/US2009/000239 (filed on Jan. 15, 2009);
(12) PCT Patent Application No. PCT/US11/27746 (filed on Mar. 9,
2011); (13) U.S. Patent Application Publication No. 2010/0029785
(published on Feb. 4, 2010); (14) Tasciotti E. et al, 2008 Nature
Nanotechnology 3, 151-157; (15) PCT Application No. PCT/US11/28861
(filed on Mar. 17, 2011); (16) PCT Application No. PCT/US11/28890
(filed on Mar. 17, 2010); (17) U.S. Provisional Patent Application
No. 61/282,798 (filed on Apr. 1, 2010); and (18) U.S. Provisional
Patent Application No. 61/322,766 (filed on Apr. 9, 2010). Each of
the above documents are incorporated herein by reference in their
entirety.
[0150] In some embodiments, the above-described microparticles or
nanoparticles within the porogen particles may allow for a release
of one or more active agents within the particles in a controlled
and sustained fashion. In various embodiments, such controlled and
sustained release may allow for optimizing a healing or
regeneration process. For instance in a case of treating a bone
defect (such as a bone fracture) the controlled and sustained
release of active agents from the microparticles or nanoparticles
may allow for optimizing the bone healing and/or regeneration
process.
[0151] The active agents which may be contained inside the
microparticles or nanoparticles of the above-described porogen
particles may include antibiotics, anti-inflammatory agents,
proteins (such as growth factor), and nucleic acids (such as DNA
and siRNA). In some embodiments, the growth factors may include one
or more of PDGF.alpha..beta., PDGF.alpha..alpha., PDGF.beta..beta.,
TGF1, TGF2, vascular endothelial growth factor (VEGF) and/or
epithelial growth factor (EGF). In some embodiments, the
microparticles or nanoparticles may contain one or more proteins,
such as fibrin, fibronectin and vitronectin. In some embodiments,
the micro or nanoparticle may contain platelet-rich plasma (PRP).
PRP may contain PDGF.alpha..beta., PDGF.alpha..alpha.,
PDGF.beta..beta., TGF1, TGF2, VEGF, EGF, fibrin, fibronectin and/or
vitronectin.
[0152] The above-described microparticles or nanoparticles within
the porogen particles may also have a variety of shapes and sizes.
In some embodiments, the maximum characteristic size of the
particles may be less than about 100 microns, less than about 50
microns, less than about 20 microns, less than about 10 microns,
less than about 5 microns, less than about 4 microns, less than
about 3 microns, less than about 2 microns, or less than about 1
micron. Yet, in some embodiments, the maximum characteristic size
of the particles may be from 100 nm to 3 microns, from 200 nm to 3
microns, from 500 nm to 3 microns, or from 700 nm to 2 microns.
[0153] Yet, in some embodiments, the maximum characteristic size of
the particle may be greater than about 2 microns, greater than
about 5 microns, or greater than about 10 microns.
[0154] A person of ordinary skill in the art will also recognize
that the shape of the microparticles or nanoparticles within the
porogen particles is not particularly limited. In some embodiments,
the microparticles or nanoparticles may be a spherical particle.
Yet, in some embodiments, the particles may be a non-spherical
particle. In some embodiments, the microparticles or nanoparticle
can have a symmetrical shape. Yet, in some embodiments, the
microparticles or nanoparticle may have an asymmetrical shape.
[0155] In some embodiments, the microparticles or nanoparticles may
have a selected non-spherical shape, such as an oblate spheroid, a
disc or a cylinder. In some embodiments, the porous particle may be
a truncated oblate spheroidal particle.
[0156] The microparticles or nanoparticles within the porogen
particles of the present invention may also comprise a porous oxide
material or a porous etched material. Examples of porous oxide
materials include, but are not limited to, porous silicon oxide,
porous aluminum oxide, porous titanium oxide and porous iron oxide.
The term "porous etched materials" refers to a material, in which
pores are introduced via a wet etching technique, such as
electrochemical etching or electroless etching. Examples of porous
etched materials include porous semiconductors materials, such as
porous silicon, porous germanium, porous GaAs, porous InP, porous
SiC, porous Si.sub.xGe.sub.1-x, porous GaP and porous GaN. Methods
of making porous etched particles are disclosed, for example, US
Patent Application Publication no. 2008/0280140.
[0157] In some embodiments, the porogen particles of the present
invention may be a nanoporous particle. In some embodiments, an
average pore size of the nanoporous particle may be from about 1 nm
to about 1 micron, from about 1 nm to about 800 nm, from about 1 nm
to about 500 nm, from about 1 nm to about 300 nm, from about 1 nm
to about 200 nm, or from about 2 nm to about 100 nm. In some
embodiments, the average pore size of the nanoporous particle can
be no more than 1 micron, no more than 800 nm, no more than 500 nm,
no more than 300 nm, no more than 200 nm, no more than 100 nm, no
more than 80 nm, or no more than 50 nm.
[0158] In some embodiments, the average pore size of the nanoporous
particle can be from about 5 nm to about 100 nm, from about 10 nm
to about 60 nm, from about 20 nm to about 40 nm, or from about 30
nm to about 30 nm. In some embodiments, the average pore size of
the porous particle can be from about 1 nm to about 10 nm, from
about 3 nm to about 10 nm, or from about 3 nm to about 7 nm.
[0159] In general, pores sizes may be determined using a number of
techniques, including N.sub.2 adsorption/desorption and microscopy,
such as scanning electron microscopy. In some embodiments, pores of
the nanoporous particle may be linear pores. Yet, in some
embodiments, pores of the nanoporous particle may be sponge like
pores.
[0160] A person of ordinary skill in the art can also envision that
various methods may be used to load active agents into the porous
particles. Methods of loading active agents into porous particles
are disclosed, for example, in U.S. Pat. No. 6,107,102 and US
Patent Application Publication No. 2008/0311182.
[0161] In some embodiments, the porous particles within the porogen
particles of the present invention are porous silicon particles. In
general, porous silicon may be bioinert, bioactive or
biodegradable, depending on its porosity and pore size. Also, a
rate or speed of biodegradation of porous silicon may depend on its
porosity and pore size. See e.g, Canham, Biomedical Applications of
Silicon, in Canham L T, editor. Properties of porous silicon. EMIS
datareview series No. 18. London: INSPEC. p. 371-376. The
biodegradation rate of porous silicon particles may also depend on
surface modification. Porous silicon particles and methods of their
fabrication are disclosed, for example, in Cohen M. H. et al
Biomedical Microdevices 5:3, 253-259, 2003; US Patent Application
Publication No. 2003/0114366; U.S. Pat. Nos. 6,107,102 and
6,355,270; US Patent Application Publication No. 2008/0280140; PCT
Publication No. WO 2008/021908; Foraker, A. B. et al. Pharma. Res.
20 (1), 110-116 (2003); and Salonen, J. et al. Jour. Contr. Rel.
108, 362-374 (2005). Porous silicon oxide particles and methods of
their fabrication are disclosed, for example, in Paik J. A. et al.
J. Mater. Res., Vol 17, August 2002, p. 2121.
[0162] In some embodiments, the porous particle may be a particle
produced utilizing a top-down microfabrication or nanofabrication
technique, such as photolithography, electron beam lithography,
X-ray lithography, deep UV lithography, nanoimprint lithography or
dip pen nanolithography. Such fabrication methods may allow for a
scaled up production of particles that are uniform or substantially
identical in dimensions.
[0163] Active Agents
[0164] As set forth above, the individual components of the
compositions of the present invention may be associated with one or
more active agents. In various embodiments, the active agent may be
associated with the biodegradable polymer matrix, the biodegradable
reinforcing particle, and/or the porogen particle.
[0165] In some embodiments, the active agent comprises antibiotics,
proteins, platelet rich plasma, cells (e.g., stem cells),
degradation inducers of porogen particles (e.g., lactic acid and/or
sodium citrate), anti-inflammatory agents, cell viability enhancing
agents (e.g., glucose), and/or imaging agents (e.g., barium
sulfate). More specific examples of active agents were described
above. Additional active agents that may be suitable for use with
the compositions of the present invention are disclosed in PCT
Application Nos. PCT/US11/28861 and PCT/US11/28890, both filed on
Mar. 17, 2010.
[0166] Use of Biodegradable Compositions as Scaffolds
[0167] In various embodiments, the compositions of the present
invention may be utilized as scaffolds. In a specific example, the
scaffolds of the present invention may be used for treating bone
defects, such as bone fractures, maxillofacial defects, and
craniofacial defects. Scaffolds of the present invention may also
be utilized for tissue engineering, tissue regeneration, and wound
healing. In additional embodiments, the scaffolds and compositions
of the present invention may be used for treating or preventing a
microbial infection, such as a bacterial infection at a site of a
bone defect.
[0168] In more specific embodiments, the scaffolds of the present
invention may be used in treating soft tissue injuries and
facilitating ligament/tendon repair. Likewise, the scaffolds of the
present invention may find various applications in tooth
regeneration, neural repair (e.g., facilitation of spinal cord
regeneration), and intervertebral disc replacement. The scaffolds
of the present invention may also be used to treat cartilage
defects. Likewise, the scaffolds of the present invention may be
utilized as vascular grafts. The scaffolds of the present invention
may also be used to make or engineer artificial tissues or organs,
such as an engineered pancreas for type I diabetic patients.
Additional applications for the scaffolds of the present invention
can also be envisioned by persons of ordinary skill in the art.
[0169] Application of Scaffolds for Treatment of Bone Defects
[0170] As set forth previously, a specific embodiment of the
present invention pertains to methods of treating a bone defect in
a subject by applying to an area of the bone defect in the subject
a scaffold of the present invention. For instance, in some
embodiments, the bone defect treatment methods of the present
invention include: (1) applying to an area of the bone defect in
the subject a scaffold that comprises: (a) a biodegradable polymer
matrix, and (b) at least one biodegradable reinforcing particle
dispersed in the matrix, as previously described. In further
embodiments, the applying step comprises injecting the subject with
a composition of the present invention. In additional embodiments,
the scaffold is formed from the composition in the body of the
subject after the injecting. In further embodiments, the scaffold
that is applied also comprises at least one porogen particle, as
described above. In further embodiments, the applied scaffold also
comprises one or more active agents, as also described.
[0171] Bone defects that can be treated with the scaffolds of the
present invention include, without limitation, bone fractures,
maxillofacial defects, craniofacial defects, spine defects (e.g.,
defects and/or injuries in intervertebral bodies), long bone
defects (e.g., weight bearing and non-weight bearing defects), and
combinations thereof. In various embodiments, the aforementioned
defects to be treated may be critical size defects and/or
non-critical size defects.
[0172] Methods of Making Biodegradable Compositions
[0173] Additional aspects of the present invention pertain to
methods of making the above-mentioned biodegradable compositions.
Such methods generally comprise dispersing in a biodegradable
polymer matrix at least one biodegradable reinforcing particle, as
previously described. Such methods may also involve the dispersion
of porogen particles and/or active agents into the biodegradable
polymer matrix. Additional details about such methods are described
in the Examples below.
[0174] Advantages and Applications
[0175] The methods and compositions of the present invention
provide numerous advantages. One of the advantages of the
compositions of the present invention may lie in the compositions'
mulitifunctionality, as the composition may have one or more of the
following properties: optimal mechanical characteristic;
injectability for irregular defects; and multiple (i.e. two or
more) stages of bioactive release to enhance the bone healing
process. The scaffold and compositions of the present invention may
also be capable of providing one or more of the following
advantages: i) cross-linking in situ, ii) conforming to a bone
geometry, iii) providing immediate mechanical stability, iv)
providing a continuous delivery of one or more active agents, which
may be, for example, antibiotics and growth factors; v) promoting
accelerated tissue regeneration and vi) degrading into benign
by-products that may be resorbed and excreted by the body.
[0176] An additional advantage of the compositions of the present
invention may be the ability to vary biodegradation and/or release
rates of various components. For instance, the present compositions
may allow for the biodegradation and/or release process to be
adjusted to match the kinetics of the bone regeneration process and
thus, progressively transfer the loads from the scaffold to the new
tissue. The development of bone architecture may be naturally
driven by the mechanical forces applied. As a result, osteoclasts
may begin resorbing bone that is not subjected to the appropriate
load and only remodel the newly formed bone in areas of high
stress.
[0177] The tunability of both the release of active agents, such as
therapeutic agents and/or imaging agents, and degradation rates of
each individual component in the scaffold may also provide the
ability to mimic and accelerate one or more natural regeneration
processes. For instance, the scaffolds and compositions of the
present invention may be designed to provide immediate stability to
a minor or substantial bone defect. The present compositions and
scaffolds of the present invention may also simultaneously initiate
and/or accelerate the natural healing cascade.
[0178] Additional aspects of the present invention will now be
described with reference to specific and non-limiting Examples.
EXAMPLES
Example 1. Scaffold Components
[0179] FIGS. 1A-1F shows alginate hydrogel microspheres
encapsulating cells and bioactive molecules. Inside of the above
porogen (.about.200-500 microns) is PRP as well as mesenchymal stem
cells (FIG. 1A-1B optical image, FIG. 1C confocal image
green-cells, FIG. 1D-1F SEM image). Also within the porogen may be
microparticles or nanoparticles, which may be coated with a
polymer, such as agarose or PLGA, that may also contain one or more
active agents loaded within the nanopores (FIG. 2).
[0180] The porogen may be dispersed within a viscous polymer
matrix, such as a PPF matrix, that may contain silica nanorods as
mechanical reinforcement (FIGS. 3A-3D). In some embodiments, the
composition with some or all mentioned components may be loaded
into a syringe and injected into the bone defect where it may
crosslink in the shape of the bone defect geometry (FIG. 4). Yet,
in some embodiments, the composition may be used for forming a
scaffold ex situ. After cross-linking, the scaffold composite may
conform to the defect geometry as seen below in FIG. 5 using a
cylindrical mold.
Example 2. Release of Active Agents from Scaffolds for
Treatment
[0181] The multifaceted nature of the injectable matrix may provide
ideal means of staggering the delivery of the above-mentioned
active agents that may enhance stem cell activity at rates
contingent upon the corresponding stage of fracture healing. For
example, PRP may provide a cocktail of all necessary growth factors
with the additional advantage of presenting them in optimal ratios
for cell growth. PRP may therefore be a supplier of bioactive
molecules throughout the entire scaffold and may be contained in
one or more components of the composition and a scaffold formed
therefrom, such as the porogen particles, the reinforcing
microparticles or nanoparticles, the microparticles or
nanoparticles with the porogen particles and the polymer matrix. In
some embodiments, PRP may be contained in more than one of the
above mentioned components of the composition or the scaffold. In
some embodiments, PRP may be contained in each of the above
mentioned components of the composition or the scaffold.
[0182] In some embodiments, one or more antibiotics may be
incorporated into one or more components of the composition and a
scaffold formed therefrom, such as the porogen particles, the
reinforcing microparticles or nanoparticles, the microparticles or
nanoparticles with the porogen particles and the polymer matrix. In
some embodiments, one or more antibiotics may be contained in more
than one the above mentioned components of the composition or the
scaffold. In some embodiments, one or more antibiotics may be
contained in each of the above mentioned components of the
composition or the scaffold. Overall, the present system may
provide antibiotics during the entirety of the healing process of a
bone defect, such as bone fracture.
[0183] The operation of the scaffold in some embodiments may be
illustrated as follows: The immediate delivery of growth factors
may be supplied through the PRP dispersed within the porogen
particles. Upon degradation of the porogen particles, the PRP may
be released into the defect site. A faster degradation (and a
faster release) may be achieved by using as porogen particles
alginate capsules with a thin layer of alginate surrounding the
encapsulated content. On the contrary, solid alginate beads may
degrade in a longer time and may therefore provide a sustained
release over 1 week or 2 weeks or 3 weeks or 4 weeks. To induce
degradation of alginate porogen particles, the reinforcing
microparticles or nanoparticles may be loaded with the calcium
chelation agent, sodium citrate, and encapsulated within the
alginate porogen itself. As the sodium citrate is slowly released
from the reinforcing microparticles or nanoparticles, the calcium
ions that gel the alginate may be replaced with citrate ions
causing the gel to disassemble and "dissolve." The rate at which
this degradation may occur may be controlled.
[0184] Secondly, upon the porogen particle degradation, the
microparticles or nanoparticles that were contained inside the
porogen particles may be released with the PRP in stage one. In
case, when these micro or nanoparticles are mesoporous silicon
microparticles or nanoparticles, they may be coated with a bulk
degrading polymer, such as PLGA or agarose, which may provide a
further extended sustained release.
[0185] In some embodiments, the mesoporous silicon microparticles
or nanoparticles may be biodegradable porous silicon with well
controlled shapes, sizes and pores. The size of the pores may
confine the space for the entrapment of an active agent of choice
while the porous silicon surface chemistry may affect the stability
and duration of its interaction with the active agent.
[0186] The ability to load active agents within the porous matrix
of the particle at room temperature may enable the use as the
active agent sensitive compounds susceptible to temperature
dependent degradation or inactivation. Polymer coating of the
mesoporous micro or nanoparticles, such as mesoporous silicon micro
or nanoparticles, may allow avoiding the burst release of the
active agent, such as an antibiotic from the pores and to achieve
its sustained release over the course of a week. Pore size and
coating strategy may be used in parallel to provide sustainable
release of active agents to enhance process of healing cascade and
to prevent the establishment of possible infections in a bone
defect site, such as fracture site.
[0187] Thirdly, for the final and longest delay of release, the
reinforcing particles embedded within the polymer matrix may
release PRP as the surrounding polymer matrix degrades exposing the
pores of the reinforcing particles to the defect site. Due to the
various rates of degradation of each above mentioned components,
the needs of each phase of the healing cascade may be met.
[0188] The composition may be a composite material having the
texture of a paste enabling it to conform to different shapes
according to the specific application and including the
reconnection of separated bones and the replacement of missing
bone. In some embodiments, the composition may be used for treating
bone defects, such as fracture or an injury for a bone in a body of
an animal, which can be a warm bloodied animal, such a mammal,
which may be a human. For example, the composition may be used for
treating a bone fracture or an injury in a human body bone, such as
a spine, a skull or a facial bone.
[0189] FIG. 6A-6F schematically depicts the use of the composition
for vertebral body compression fractures. PPF refers to the polymer
matrix, which may be poly(propylene fumarate); MSN refers to
reinforcing micro or nanoparticles, which may mesoporous silica
nanorods; SE refers to micro or nanoparticles inside the porogen
beads, such micro or nanoparticles may be mesoporous silicon micro
or nanoparticles.
Example 3. Synthesis, Characterization and Use of Alginate
Porogens
[0190] The following example provides steps for incorporation of
cells and platelet-rich plasma (PRP) and bioactive molecules into
an alginate microsphere matrix during a fabrication process.
[0191] Protocol for Synthesis of Alginate Porogen Microparticles
with the Incorporation of Cells and Platelet-Rich Plasma
[0192] As depicted in FIG. 7C-7E, Calcium alginate beads were
synthesized by emulsion in mineral oil with low surfactant
conditions and acetic acid as a catalyst. In order to optimize the
process and accomplish beads with sizes ranging from 300-500 .mu.m,
the concentration ratio of sodium alginate and platelet-rich
plasma, the amount and type of surfactant, the stir rate and size
of beaker and stir bar used for creating an optimal volume were all
varied within the same protocol. Select runs of this process are
provided in Table 1 below.
TABLE-US-00001 TABLE 1 Runs 1-6: Variables manipulated, values
employed, bead size range obtained. Percent Surfactant Alginate
Starting pH of Concentration Bead Size Range Run Solution Alginate
Solution (Span80) Stir Speed (average) Notes 1 2 7.55 2% 2 on VWR
80-100 .mu.m Did not sonicate CaCO3 prior to addition to alginate.
Added CaCl2 to alginate mineral oil mixture 2 2 7.55 4% 2 on VWR
80-200 .mu.m Added Alginate- mineral oil mixture to CaCl2 3 5 7.65
4% 5-6 on Cimarec 80-250 .mu.m 4 5 7.61 4% 4 on Cimarec 80-300
.mu.m Larger beads but increased clumping (as seen in 4b) 5 5 7.58
3% 5-6 on Cimarec Poor shape, difficult to characterize 6 4 7.56 3%
4 on Cimarec 80-300 .mu.m Some clumping, a few beads in 300 .mu.m
range
[0193] The addition of PRP into the alginate matrix was found to be
essential to create a desired viscosity for bead creation within
the aimed 200-500 microns. The difference in size is illustrated in
FIGS. 7A-7B and 7F.
[0194] The protocol for the incorporation of live cells into the
microparticles is as follows: 5 grams of sodium alginate was slowly
dissolved in 75 mM NaCl/12.5 mM HEPES in PRP:D1 water (1:1) (5%
w/v). The pH of the solution was adjusted to a value of -7.5.
Sonicated aqueous CaCO3 mixture with 500 mM Ca2+ equivalent was
then added to the alginate solution. Mesenchymal stem cells were
then added to the alginate solution suspended in PRP. The
alginate-cell-CaCO3 mixture was then added to a solution of mineral
oil with 2 Span80 by volume and stirred with a magnetic stir bar
for 15 minutes. With continued stirring, a mixture of mineral oil
and glacial acetic acid was slowly added and allowed to mix for 10
additional minutes to initiate the release of calcium from the
carbonate and the subsequent gelation of the calcium alginate
beads. Beads were then separated from the oil dispersion by
partitioning the mixture above into an aqueous CaCl.sub.2 solution.
The beads were then collected by pipette and washed on a vacuum
filter with 1% Tween 80 to remove residual oil. Due to the size
distribution, a method of sieving out unwanted sizes was developed.
Briefly, a 500 micron ASTM sieve was used to remove all beads
larger than 500 microns, and a 212 micron sieve was used to remove
all beads smaller than 212 microns.
[0195] Characterization of Alginate Porogens
[0196] FIG. 1A provides optical images of alginate porogens with
PRP (top left), and alginate porogens with mesenchymal stem cells
(FIG. 1B, top center). FIG. 1C also shows confocal microscopy
images of alginate porogens with mesenchymal stem cells labeled
with green fluorescence (CSFE) (top right). In addition, FIGS.
1D-1F shows SEM images of alginate porogens (bottom).
[0197] Alginate porogens were fabricated using the above-mentioned
protocol and were characterized by optical microscopy (FIG. 1A-1B)
(Nikon Eclipse TS 100), confocal microscopy (FIG. 1C) (Leica MD
6000), and scanning electron microscopy (SEM) (FEI Quanta 400 ESEM
FEG) (FIGS. 1D-1F). The samples were sputtered with 20 nm gold by a
Plasma Sciences CrC-150 Sputtering System (Torr International, Inc)
prior to SEM analysis.
[0198] FIGS. 1B-1C (top center and top right) also includes images
of mesenchymal stem cells stained with a fluorescent dye. Calcium
alginate beads were synthesized by the emulsion process described
above with the incorporation of cells into the aqueous alginate
phase. The stem cells were stained with the green fluorescence dye
Carboxyfluorescein diacetate, succinimidyl ester (CSFE) using a 25
.mu.m staining solution. After 12 hours of incubation
post-staining, the cells were trypsinized and re-suspended in the
5% alginate/PRP solution at 2.times.10.sup.6 cells per ml. The
emulsion was then performed as described above, thereby creating
alginate and PRP beads encapsulating live cells. Beads with cells
were then incubated in DMEM complete media (10% FBS, 1% antibiotic)
at 37 degrees Celsius prior to imaging.
[0199] Enhancing the Mechanical Properties of a Porous Polymer
Matrix Through Incorporation of Alginate Porogens
[0200] PPF and alginate scaffolds were created using Teflon molds
with dimensions 6 mm d.times.12 mm h. Briefly, the PPF monomer was
diluted with N-vinyl-2-pyrrolidone (NVP) using a 1:4 ratio prior to
dispersion. A mixture of 40% alginate porogens by weight within PPF
was mechanical stirred. 20 mg of benzoyl peroxide (BP) was then
added to initiate the cross-linking of PPF along with
N,N-Dimethyl-p-toluidine (DMT) to accelerate the reaction. The
mixture was then poured into the Teflon mold and placed at 60
degrees Celsius overnight to fully cross-link.
[0201] The compressive mechanical properties of the
alginate-incorporating constructs were measured according to
IS05833 standards. 6 mm.times.12 mm cylindrical scaffolds (n=5)
incorporating 40% alginate microspheres by weight were compressed
along their long axis using a mechanical testing machine with a 10
kN load cell (MTS). As a comparison, 80% salt PPF scaffolds of
equivalent size were created and the salt leached out to create a
porous structure. The 80% salt porous scaffolds were then tested
using the same methods described above. The young's modulus and
stress at offset yield were recorded and are illustrated in FIGS.
8A-8B, respectively.
[0202] Results from MTS Testing Showing Mechanical Reinforcement
Due to Presence of Alginate Microparticle Porogens
[0203] As summarized in FIGS. 8A-8B, the alginate porogens provide
an 8-fold increase in mechanical strength compared to
pre-fabricated PPF porous scaffolds by temporarily filling the
voids until they undergo biodegradation. Furthermore, the elastic
modulus of the porogen composite provides a significantly closer
match to that of trabecular bone within the vertebral body (165-291
MPa).sup.1 than the current PMMA standard (48-76 MPa).sup.2.
[0204] The addition of calcium alginate porogens into the (PPF)
based matrix renders the temperature increase, from the exothermic
cross-linking reaction, virtually undetectable. This may alleviate
existing concerns with current injectable polymers of damaging
surrounding neural and vascular structures.
[0205] FIG. 9 presents temperature profile of PPF cross-linking
with varying alginate porogen content. Calcium alginate beads were
synthesized by emulsion/internal gelation methods described above
without the addition of cells. The change in temperature of the
scaffold during cross-linking was measured by recording the
temperature of the mixture as a function of time after the addition
of the last component. The mixture was packed into a Teflon
cylindrical mold (According to IS05833 for acrylic resin cements)
where a temperature probe connected to a multimeter was positioned
at the center of the mold to record the temperature of the mixture
every 1 second until the temperature began to drop and then
stabilize.
[0206] Controlled Release of Growth Factors from Platelet Rich
Plasma to Induce Cell Migration, Proliferation and
Differentiation
[0207] Alginate beads with platelet-rich plasma were fabricated and
separated into three size ranges using various sieves (x<212
.mu.m, 212<x<500 .mu.m, and x>500 .mu.m). 100 mg of
swelled alginate beads of each size range were weighted out into
eppendorf tubes and 200 ul of DMEM was added to each sample.
Samples were then placed horizontally on a rotator at 37 degrees
Celsius. At various time point samples were spun down (2500 rpm for
5 min) and 100 .mu.l of supernatant was removed from each sample
and stored at -20 C for later ELISA analysis.
[0208] The release of PDGF, VEGF, and RANTES from PRP with alginate
porogens of varying sizes is demonstrated in FIG. 10A. The effect
of the mitogenic growth factors, PDGF-AB/BB/AA on cell
proliferation is demonstrated in FIG. 10B. Similarly, the
stimulatory effect of RANTES release from the complex is
illustrated in FIG. 10C. Furthermore, the induction of angiogenesis
was been confirmed in a Lewis Rat subcutaneous implantation of the
PRP/alginate porogens (FIG. 10D).
[0209] Cryopreservation of Biological Components
[0210] As shown in the cell viability assays of FIG. 10E, porogen
particles also provide cell viability protection during the
injecting and cross-linking process of the polymer scaffold.
Furthermore, as demonstrated in FIG. 10F, The degradation of the
porogen can be artificially controlled through the controlled
delivery of calcium chelation agents that cause dissolution of the
porogens.
Example 4. Synthesis and Characterization of Silica Nanorods
[0211] The following experiments pertained to porous silica nanorod
fabrication with desired aspect ratios for mechanical
reinforcement. In the adjustments of reagents such as ammonia,
CTAB, TEOS and 3-(trimethoxysilyl)propyl methacrylate silane, the
aspect ratio of the silica may be increased to the desired size for
nano reinforcement. In addition, mesoporous silica nanorods may
contain one or more active agents (including but not limited to,
contrast agents, metallic ions, fluorescent dyes, and cations),
which may be incorporated into porous silica nanorod matrices
during the fabrication process. The following protocols also
describe surface modification methods to covalently bond the porous
silica nanorod to the backbone (or side chains) of polymer matrices
to be reinforced.
[0212] Protocol for Synthesis of Silica Nanoparticles with
Incorporated Barium Sulfate
[0213] CTAB was dissolved in 70 ml H.sub.2O for 30 minutes.
Ammonium hydroxide was added and the mixture was stirred vigorously
for 1 hr. TEOS and Barium Sulfate (70 mol % TEOS) (optional) was
added and stirred for 4 hr. The solution was centrifuged at 13,000
rpm for 10 min and washed in a mixture of ethanol and water several
times. The particles were dried under vacuum overnight at room
temperature. The surfactant was removed by placing the dried
particles in 100 ml ethanol and 1 ml concentrated HCl for 6 hours.
The solution was centrifuged at 13,000 rpm for 10 min and washed in
a mixture of ethanol and water several times. This washing process
allows for removal of surfactant and the survival of the oxidized,
active surface. The molar ratio of the reaction was 100 TEOS: 29
CTAB: 35,700 H.sub.2O: 714 NH.sub.3--H.sub.2O (varies with desired
size/aspect ratio). Size and morphology were characterized by
dynamic light scattering (DLS) transmission electron microscope
(TEM), and scanning electron microscope (SEM). Surfactant removal
was characterized through zeta potential and FTIR. Pore size and
volume were observed through BET. The results are summarized in
Table 2 below and depicted in FIGS. 11A-11C and FIGS. 14-16.
TABLE-US-00002 TABLE 2 Increase in aspect ratio and decrease in
size through adjustment of TEOS, CTAB and Ammonia. CTAB H.sub.2O
NH.sub.3 TEOS 1 0.4 (567 mg) 1000 (70 ml) 20 (3.03 ml) 2.8 (2.41
ml) 2 0.8 (1134 mg) 1000 (70 ml) 20 (3.03 ml) 2.8 (2.41 ml) 3 0.4
(567 mg) 1000 (70 ml) 10 (1.51 ml) 0.7 (0.6025 ml)
[0214] Protocol for Synthesis of Silica Nanoparticles with the
Incorporation of Acrylate Surface Modification for Better
Incorporation into Polymer Matrix
[0215] Nanorods were also synthesized in the same manner with the
inclusion of 3-(trimethoxysilyl)propyl methacrylate (3.5 mol %
TEOS) with TEOS and Barium Sulfate. The molar ratio of the reaction
was 100 TEOS: 14 CTAB: 142,000 H.sub.2O: 1428 NH.sub.3--H.sub.2O.
The results are illustrated in FIGS. 11A-11C.
[0216] Protocol for the Post-Synthesis Modification of Silica
Nanorods
[0217] 20 .mu.l of millipore water was added to 1 mg of particles
and sonicated for 10 minutes. A solution of 2.04% acrylate silane
and 3.06% Millipore water (optional percentages) in IPA was
prepared (980 .mu.l). The solution was mixed at 35.degree. C. at
1300 RPM for 2 hours. After modification, the particles were
centrifuged at 13000 rpm for 10 minutes. The supernatant was then
removed and replaced with 100% anhydrous IPA for washing. This step
was repeated two more times. The supernatant was then removed and
the particles were moved to a vacuum oven overnight at 60.degree.
C. The results are depicted in FIG. 13.
[0218] Characterization of Two Forms of Silica Synthesis and
Modification and Barium Incorporation
[0219] SEM and TEM images of silica nanorods are presented in FIG.
3, FIGS. 11A-11C and FIGS. 14-16. Particle morphology of these
materials was determined by scanning electron microscopy (SEM)
using a (FEI Quanta 400 ESEM FEG). with 10 kV accelerating voltage
and 0.005 nA of beam current for imaging. For transmission electron
microscopy (TEM) studies, a small aliquot was taken from a
suspension of isopropal alcohol and placed in a lacey carbon-coated
TEM grid, which was pulled through the suspension and allowed to
dry in air. The resulting sample was examined with a Philips model
CM-30 TEM operated at 300 kV.
[0220] BET (Brunauer-Emmett-Teller) of silica nanorod pore size
distribution is presented in FIGS. 19A-19B. The median pore
diameter were measured using N.sub.2 adsorption/desorption
measurements in a Micromeritics ASAP 2000 BET surface analyzer
system. The data were evaluated using the Brunauer-Emmett-Teller
(BET) and Barrett-Joyner-Hal-enda (BJH) methods to calculate the
size distribution.
[0221] Biocompatibility of Silica Nanorods
[0222] Biocompatibility data for silica nanorods is presented in
FIGS. 20A-20C. Acrylate modified silica nanoparticles were
dispersed in complete media at a concentration of 0.01 mg/ml. This
media was added to MDA 231 cells and allowed to incubate for 8 and
24 hours. After incubation, the media was removed and saved. The
cells were then washed with PBS twice, removed from the wells with
trypsin, and placed in the corresponding Eppendorf tube containing
the original media. Additional fresh media was added to the tubes
to stop trypsin activity. The tubes were then centrifuged in at
1,500 rpm for 4 minutes to remove the media. The cells were then
washed with Annexin Binding Buffer and centrifuged at 1,500 rpm for
4 minutes to remove the buffer. The samples were then stained with
Annexin V conjugated with Alexa Fluor 488. Annexin V is a protein
that attaches to phosphatidylserine on the outer surface of the
cell only during apoptosis. Staining with Annexin V conjugated with
Alexa Fluor 488 will cause apoptotic cells to be fluorescent. The
samples were then analyzed for viability using a flow
cytometer.
[0223] Acrylate modified silica nanoparticles were assessed for
biocompatibility. The silica nanoparticles are biocompatible at the
concentration of 0.01 mg/ml as seen by the greater than 90%
similarity of viability compared to the control cells not incubated
with silica nanoparticles.
[0224] Next, contrast agents were incorporated to enable the
monitoring of the scaffold degradation and tissue infiltration.
[0225] X-Ray Detection of Barium Sulfate in Loaded Silica
[0226] Agarose scaffolds containing Barium Sulfate loaded silica of
0, 2.5 and 5% silica were prepared. Low melt agarose (2.3%) was
mixed with barium sulfate loaded silica dispersed in 400 .mu.l of
H.sub.2O. The mixture was subsequently loaded into cylindrical
molds (6 mm.times.20 mm) and placed in ice for one hr. The agarose
rods were place in a high resolution x-ray and read at 85 kV to
show the ability to track silica and scaffold degradation through
imaging. FIG. 21 shows X-ray image of agarose composite with from
right to left 0%, 0.5% and 2% silica nanorods containing barium
sulfate. FIG. 22 shows GIF (Gatan Energy Filter) image through TEM
showing presence of barium and sulfur within silica nanorods.
[0227] Protocol for Loading and Releasing Bioactive Agents into
Silica Nanorods
[0228] Weigh out 10 mg of dexamethasone and place into eppendorf
tube. Add 20 .mu.l of solvent and dissolve the dexamethasone. Add
the dexamethasone solution to 1 milligram of particles. Disperse
the silica in the solution and place in the thermomixer for 1 hour
at 35.degree. C. Centrifuge the particles and remove the
supernatant and place it into a labeled tube for loading amount
determination. Dry particles. Wash particles before using.
[0229] Controlled Release of Glucose, Antibiotics, and
Anti-Inflammatory Drugs
[0230] FIG. 23 is a graph demonstrating controlled release of model
drug, DOX-HCl, from silica nanorods. FIG. 24 is a graph
demonstrating controlled release of Cefazolin from silica nanorods.
Next, the porous silica nanorods were dispersed within a polymer
matrix.
[0231] Nanocomposite Fabrication
[0232] PPF was mixed in NVP in a 1:2 mass ratio. Silica nanorods
(co-condensated and post modified) were then mixed into the polymer
blend at loading concentrations of 2.5, and 5 wt %. The
cross-linking initiator, bp, was prepared in a 0.1 g/mL NVP
solution and added to the composite mixture at 0.5 wt %. Samples
for compressive testing were prepared by pouring the nanocomposite
mixture into Teflon molds (6.5 mm diameter, 40 mm length). Samples
were subjected to vacuum to remove air bubbles within the polymer
and then placed in the oven at 60 degrees. Once dried, samples were
cut using a diamond saw into compression testing bars of
approximately 6.5 mm diameter and 13 mm height.
[0233] Enhancing the Mechanical Properties of Polymer Matrix
through Incorporation of Silica Nanorods
[0234] Mechanical properties of solid nanocomposite samples were
determined by an 858 Material Testing System mechanical testing
machine (MTS System Corporation, Eden Prairie, Minn.) with a sample
size of five for each group (except for comparison studies with
mixed and silica nanorod composites which were conducted with a
sample size of three). Compressive mechanical testing was conducted
in accordance with ASTM D695-95. Cylindrical samples were placed
between two plates as the cross-head lowered onto the sample at a
constant rate of 1 mm/min until failure. The cross-head was lowered
at a rate of 10 mm/min to the center of each specimen until
failure. Force and displacement measurements were recorded and
converted to stress and strain based on sample dimensions. The
compressive modulus was calculated as the slope of the initial
linear region of the stress-strain curve. Compressive fracture
strength was calculated as the maximum stress applied prior to
failure.
[0235] FIG. 25A shows stress offset of control, 2.5% co-condensated
silica nanorods (CC) and post-modified silica (E). FIG. 25B shows
compressive modulus of 2.5% co-condensated silica nanorods (CC) and
post-modified silica when dispersed in PPF polymer.
[0236] Enhancing Cell Viability Through the Controlled Release of
Glucose into the External Environment of the Scaffold.
[0237] Glucose Lactam loading and release was determined as
follows. 18.81 mg of 2-keto-D-glucose was weighed out and placed
into an eppendorf tube. 250 .mu.L of water was added to dissolve
the glucose (final concentration: 75.24 mg/mL). 20 .mu.L of the
2-keto-D-glucose solution was added to 1 milligram of particles.
The silica was then dispersed in the solution and placed in the
thermo-mixer for 1 hour at 35.degree. C. The particles were then
centrifuged. The supernatant was removed and placed into a labeled
tube for loading amount determination. The particles were then
washed twice with water-save the supernatants and dried for 1 hour
in lyophilizer To release the glucose lactam from the silica, the
particles were rotated and incubated at 35.degree. C. and
centrifuged. The supernatant was removed at 1 hr, 2 hr, 3 hr, 6 hr,
8 hr, 12 hr, 24 hr, 48 hr etc. 200 ul of fresh PBS was then added
to the particles and the particles were re-suspended and placed
back on the rotator at 35.degree. C. The supernatant at the
different time points was then read through a spectrophotometer and
measured against a standard curve of glucose lactam to understand
the quantity of glucose lactam released.
[0238] Enhancing the Osteoconductive Properties of Composite
Scaffold Materials
[0239] FIG. 26A presents mineralization data showing increase in
Calcium and phosphate in the presence of silica. In the sample of
the agarose substrate in osteogenic media, where silica
nanoparticles are present, there was a higher amount of calcium and
phosphate deposited on the surface of the agarose compared to that
of the control where the silica nanoparticles are not present (FIG.
26B)
Example 5. Development of Injectable Composite Putty
[0240] Simultaneously, we have developed an injectable scaffold
featuring in situ setting polymer matrices with alginate-bead based
porogens. Through the use of porogens within the putty matrix, the
cells, and bioactive molecules loaded within degradable
microparticles can be delivered slowly in vivo by the degrading
porogen while simultaneously creating pores of optimal size for
tissue and blood vessel infiltration. The porogen technique
involves dispersing particles, such as hydrogel microspheres in a
matrix of scaffold material [7]. After the scaffold material
hardens, a composite consisting of the porogen and polymer remains
and complete dissolution of the porogen can occur in vivo over
time. The end result is a 3D porous scaffold. The porogens will
therefore serve three key functions: (1) provide immediate
mechanical stability to the scaffold (2) protect and delivers cells
and biological molecules essential for accelerating the
regenerative process and (3) create pores within the scaffold
post-injection to invoke the infiltration of natural bone. The
porogens can be tailored to control the pore size and porosity of
the scaffold. The size of the porogen sphere determines the size of
the pores within the scaffold and the polymer to porogen ratio is
what determines the scaffold's porosity. The developed porogen will
encapsulate the growth factor-releasing nanoporous silicon
enclosures (NSE), bioactive PA, MSC, and a nutrient rich cocktail
of PRP prior to injection. Once injected, the physiological fluid
will degrade the porogen thereby releasing the contents into the
surrounding environment and creating a porous structure.
[0241] The alginate beads were combined with PPF to form a
composite putty capable of in situ cross-linking after injection
into a fracture site. While PPF cross-linking causes significantly
less exothermic heating than PMMA, we supplemented the PPF phase
with pre-cross-linked (but still chemically viable) PPF
microparticles to serve as a "heat sink" and minimize exothermic
reactions in vivo. It was found that by including pre-polymerized
PPF particulates into the putty, peak temperature reached only
10.degree. C. above body temperature for a duration of 1 to 3
minutes (FIG. 26C). The addition of 15% pre-cross-linked PPF
dampened the temperature increase by half. Peak temperature
occurred at 1 minute 45 seconds. An increase of 5-6.degree. C.
within the bone is comparable to a fever and biologically
acceptable. Mechanical testing that followed indicated no loss in
compressive strength due to using pre-polymerized PPF.
[0242] To test the compressive strength of the putty, PPF and
PPF-alginate bead composite scaffolds were fabricated in
cylindrical Teflon molds. It was observed that the addition of
porogen beads significantly increased the compressive modulus of
the material compared to porous scaffolds and closely matched that
of trabecular bone, the intended tissue for regeneration (FIG.
8A).
[0243] In sum, mesoporous silicon nanorods can incorporate the
following five properties: 1) mechanical reinforcement of the
polymer matrix; 2) delivery of active agents, such as bioactive
molecules; 3) mineral deposition, 4) increase in cell viability and
5) provide imaging/monitoring capability through incorporation of
imaging agents, such as contrast agents into the porous silica
nanorod.
Example 6. Surface Functionalization of Mesoporous Particles for
the Sustained Delivery of Antibiotics for Orthopedic
Applications
[0244] The present inventors discovered that functionalization of a
surface of porous or mesoporous particle with a polymer may provide
for a sustained release of an active agent, such as a therapeutic
and/or imaging agent, contained in the particle's pores.
Summary and Background
[0245] Bacterial infection is one of the most common problems after
orthopedic implant surgery. If not prevented, bacterial infection
may result in serious and life threatening conditions, such as
osteomyelitis, which has shown a great necessitate for local
antibiotic delivery systems in the treatment of infections.
Mesoporous silicon (MPS) with antibiotics may be one of the
relevant approaches for obtaining a controlled drug release. To
characterize MPS, surface charge, surface modification and size
distribution, and in vitro antibiotic release from them were
carried out. HPLC and UV spectroscopy were used for the assay of
two different antibiotics: Cefazolin and Clindamycin sodium and the
assays method were validated. MPS with 10-100 .mu.L diameter having
200 nm in length obtained by etching technique and sorted by
centrifugation are used in this study as novel drug delivery. It
has shown that surface modification of MPS leads to decelerating
the release of the integrated antibiotics. As well,
biodegradability of MPS in phosphate buffer saline (PBS) solution
was demonstrated. Such antibiotic release from the MPS may provide
more reliable antibiotic protection at a targeted site of a bone
defect.
[0246] Despite particular treatment, open fractures (broken bones
in communication with the environment) present high rates of
complications because of the risk of bacterial infections and
chronic osteomyelitis that can threaten the viability of the limb
and even the life of the patient. Standard care for open fractures
requires irrigation, debridement, stabilization, and antibiotic
therapy and often results in multiple procedures according to the
severity of the wound and the onset of infections.[1]
[0247] The lack of proper control over a drug release rate and
target delivery area is a huge disadvantage for conventional drug
tablets. Tablets tend to provide rapid and immediate release of
therapeutic agents and require more frequent and repeated dosages
for maintaining therapeutic levels, causing unwanted fluctuations
in drug amounts delivered to the blood and tissue. In order to
circumvent problems in drug adsorption, metabolism, and irregular
concentrations and to optimize the therapy itself, a controlled
release dosage is advantageous over conventional tablets.
Biomaterials with nanoscale features have become increasingly
popular as controlled release reservoirs for drug delivery.
Nanoscale drug delivery systems may be able potentially tune
release kinetics, enhance availability and distribution over time,
and minimize toxic side effects, thus increasing the therapeutic
effect of a given drug. Localization, controlled release, and
sustainability of drugs over long periods of time within the body
may be some of the challenges in the design of effective drug
therapies.
[0248] Delivery systems able to release antibiotics over an
extended period of time may solve all these issues and provide
efficacious alternative solutions to the current approaches. The
objective of this study is to prove that MesoPorous Silicon (MPS)
may be effectively used in combination with orthopedic implants
and/or with scaffolds for bone tissue engineering to reduce the
onset of infections and to enhance the ability of bone to heal in a
timely fashion. MPS may offer significant advantageous properties
for drug delivery applications as it favorably extend drug
pharmacokinetics, stability as well as bio-absorbability.
[0249] Biodegradable MPS with well-controlled shapes, sizes and
pores have been developed. [2] The size of the pores may confine
the space for the entrapment of the antibiotic of choice while MPS
surface chemistry may affect the stability and duration of its
interaction with the antibiotic. The size of the pores and the
surface chemistry can be easily altered and controlled to tune
release kinetics. The ability to load drugs within the porous
matrix of the particle at room temperature enabled the use of MPS
also with sensitive compounds susceptible to temperature dependent
degradation or inactivation.
[0250] Mesoporous Silicon Fabrication
[0251] Porous silicon fragments were produced by fractionation of
sonicated multilayer porous silicon films. The multilayers were
produced by anodic etch of a 100 mm p++Si wafer in a 1:2 HF:Ethanol
solution. A 5A current was applied for 2-6 s followed by a 2 A
current for 20 s. The two step process was repeated for 30 cycles
with a stop of 8 s in between each cycle. Finally a release current
of 7 A was applied for 5 s. The wafer was rinsed in DI water and
briefly sonicated in isopropanol to detach the porous layer. The
porous silicon suspension in isopropanol was transferred to a glass
bottle and sonicated for 24 hours to reduce average fragment size.
Successive centrifugation steps fractionated the obtained porous
silicon fragments. Initial centrifugation at 4300 rpm sedimented
the micron and supra-micron fraction. The supernatants were
transferred to Oak Ridge Teflon Centrifuge Tubes, and centrifuged
at 10 K.times.g RCF using a Beckman Ultracentrifuge to sediment the
sub-micron fraction. The supernatants were centrifuged again at
26K.times.g RCF to sediment the low sub-micron fraction, while the
nanometric fraction remained suspended and kept in solution. After
centrifugation, fragments were fractionated into micron,
sub-micron, low sub-micron, and nanometer ranges. We characterized
each production lot by SEM verifying their compliance to the
required standards. The fragments were oxidized in hydrogen
peroxide solution.
[0252] Particle Size Distribution
[0253] 5 .mu.L of resuspended 20 ml of Isopropanol mixture of MPS
in solution was diluted in 10 mL double filtered Isotonic solution,
ultrasonicated for a few seconds, and subjected to inverting for
few times before measurement to achieve well mixing. MPS were then
sized using a Beckman Mutisizer IV. Triplicate analyses were made
on each suspension, which corresponded to a single batch. Results
are expressed as the mean MPS diameter (mm) of the three batches as
a function of volume (%).
[0254] MPS Surface Charge
[0255] 5 .mu.L of resuspended 20 ml of Isopropanol mixture of MPS
in solution was washed and diluted in 1400 .mu.L 10 mM 7.4 pH
Phosphate buffer, ultrasonicated for a few seconds, and subjected
to vortexing for 5 minutes to prevent aggregation. MPS were then
analyzed using a Brookhaven Zeta potential analyzer. Triplicate
analyses were made on each suspension, which corresponded to a
single batch. Results are expressed as the mean MPS surface charge
of the three batches.
[0256] MPS Surface Modification
[0257] The mesoporous silicon were transfer to premeasured
ultra-centrifuge tubes. They were spanned down using Beckman
Coulter Ultracentrifuge at 12000 RPM for 20 min at 4.degree. C. The
supernatant of each vial was removed and stored separately. The
fragments were dried out using vacuum oven for approximately 2-4
hour depends upon volume of the fraction at 75-80.degree. C. The
mass of the dried fragments was measured before proceed to
oxidation step. 4 mL of H.sub.2O.sub.2 were Added to each tubing
and shaked for a few times by hand and left for 2-3 hours. Each
sample was sonicated for 1-2 minutes. The sonicated sample was
placed in the oven set at 90.degree. C. for 2 hours to be
completely oxidized. Isopropanol alcohol (IPA) was added to cover 1
cm above the height of the dried-out fragments level. The samples
were washed 3.times. with IPA.
[0258] Loading of Antibiotics
[0259] At room temperature (25.degree. C.), the MPS samples were
placed in a vacuum (10-4 Torr) for approximately 20-30 min to rid
nanopores of any trapped alcohol. The high concentration antibiotic
solution loaded was 1 mg/mL of each antibiotics (from Sigma
Aldrich). The samples were incubated for 2 hours to allow
sufficient time for the drug to fully penetrate into pore structure
and then the drug-loaded MPS samples were washed two times with
phosphate-buffered saline (PBS), pH 7.2 (GIBCO).
[0260] Agarose coated MPS
[0261] 5 mg and 10 mg of agarose (Sigma) were reconstituted into 1
mL of deionized water respectively and the well-mixed powder was
melted at 65 C for 20 minutes and cool down to 37 C. Then, 20 .mu.L
of agarose solution was added 20 uL of fragments loaded, suspended
and sonicated. The samples were mix and stored in the thermo-shaker
for 15 min. The samples were centrifuged down (10 min; 14000 rpm;
37 C) and the supernatant was collected while the solution was
still warm. Then, the samples were resuspended in deionized water
and sonicated for few min.
[0262] Gelatin Coated MPS
[0263] All MPS were coated by modified hot-melt method. The
well-mixed gelatin powder was melted at 65 C and brought to 37 C.
The mixture was then diluted into two concentration solutions, and
cooled at room temperature. The resulting coated MPS were washed
and dried in vacuum.
[0264] Release Studies
[0265] MPS samples were individually incubated in a humidified 95%
air/5% v/v CO2 incubator at 37.degree. C. in 500 .mu.L of fresh
PBS. At designated time points, 500 gL of the release medium was
exchanged and the antibiotic concentration was determined as
described below.
[0266] Quantification of Antibiotic Concentrations
[0267] Both drugs have characteristic spectra by UV-VIS
(ultraviolet and visible light) absorption spectroscopy with peaks
at 210 nm and 270 nm for Clindamycin and Cefazolin, respectively.
With drug standards ranging from 1 to 200 .mu.g/mL, absorbance
calibration curves obtained at these peak wavelengths gave linear
graphs with correlation coefficients greater than 0.98. High
performance liquid chromatography (HPLC) methods were used to
further investigate Clindamycin and Cefazolin release from MPS.
HPLC was performed with a Hitachi chromatography system with
LaChrom software control. The chromatography system used a Agilent
Technologies Zorbax Eclipse Plus C18, a 50-.mu.L injection volume,
detection at 210 nm and 270 nm and a mobile phase was composed of
0.05M Monobasic Potassium Phosphate: Acetonitrile: Tetrahydrofuran)
(76.5:23.0:0.5, v/v/v), at a flow rate of 1 mL/min. Calibration
graphs were linear in the 1-200 .mu.g/mL concentration range. A
relatively good resolution of Clindamycin peak from interferences
was achieved at retention time between 1.3-1.5 min.
[0268] Scanning Electron Microscopy (SEM) Analysis
[0269] MPS were observed by scanning electron microscopy. Samples
were washed with ethanol. Specimens were mounted on SEM stubs (Ted
Pella, Inc.) using conductive adhesive tape (12 mm OD PELCO Tabs,
Ted Pella, Inc.). Samples were sputter coated with a 10 nm layer of
gold using a Plasma Sciences CrC-150 Sputtering System (Torr
International, Inc.). SEM images were acquired under high vacuum
condition, at 20 kV, spot size 3.0-5.0, using an FEI Quanta 400 FEG
ESEM equipped with an SE detector.
[0270] Results and Discussion
[0271] The release of antibiotics from non coated MPS was
characterized by a 30% burst within the first day (FIG. 27) and
subsequent release of remaining antibiotics within 4-6 days. In
contrast, surface coated MPS released only 10-15% within first day.
Substantial release was completed within 6 days. Bare MPS controls,
without any surface modification, showed 60-70% antibiotic release
within 1-2 days as expected. This proved that the nanostructures of
MPS pores were controlling the sustained drug release. This shape
of release profile was similar for both antibiotics from MPS.
Nevertheless, a near sustain drug release was achieved over 5-6
days with an average release rate over all the time points was
400-500 .mu.g. The desired release profile for many drugs would
follow this type of sustained release so that the drug levels in
the body remain constant while the drug is being introduced.
[0272] FIGS. 28 and 29 illustrate the accumulative release profile
of Cefazolin within 5-6 days from MPS agarose and APTES coated,
respectively. MPS matrix degradation over time was evaluated with
flow cytometric analysis and multisizer analysis, as shown in FIGS.
30A-30C and 31, respectively. FIGS. 32 and 33 show FACS analyses of
the MPS. FIGS. 34A-34B present zeta potential of differently
surface modified MPS.
[0273] Morphological Changes
[0274] To clarify the release mechanism, MPS morphology was studied
by SEM during course of release. The images of MPS matrix loaded
with antibiotics have been showing significant dissolution of the
drug due to the porosity and surface degradation of the MPS matrix
nanostructure which can be tailored for some biomedical
applications.
[0275] In our MPS delivery system and as it has been suggested by
others, an active carrier system can sometimes be a part of an
additional treatment in terms of contribution to the healing of the
surrounding environment tissue. Another benefit of silicon
degradation byproduct is that it is non-toxic. Cefazolin and
Clindamycin are few examples of common pharmaceutical antibiotics
that reduce the bacteria biofilm formation which were used as a
model drug for this study.
[0276] Current advanced drug delivery improves delivery efficiency
and localization which may directly reduce prescribed dosages to
the patient. In medical practice, antibiotics are given in large
dosages but, controlled sustained release, would help reduce the
toxic side effects, drug waste, and additional complications. In
addition, the sustained release from MPS may be tailored to provide
the correct therapeutic dose to avoid adverse effects. Other
properties, such as interactions between drug and matrix, pore
size, pore geometry, and matrix reactions with surrounding media
are just a few other aspects needed to be considered for controlled
drug delivery system design.
REFERENCES
[0277] [1] Starr A J. J Bone Joint Surg Am. 2008 February: 90 Suppl
1:132-7. [0278] [2] Tasciotti E, Liu X, Bhavane R, Plant K, Leonard
A D, Price B K, Cheng M M, Decuzzi P, Tour J M, Robertson F,
Ferrari M. Nat Nanotechnol. 2008 March; 3(3):151-7. Epub 2008 March
[0279] [3] Chiappini, Ciro., Tasciotti, E., Fakhoury, J. R, Fine,
D., Pullan, L., Wang, Y C, Fu, L., Liu, X, Ferrari, M. J
ChemPhysChem. [In Press]. [0280] [4] Vallet-Regi M. 2006. Chem Eur
J 12:5934-5943. [0281] [5] Horcajada P, Ramila A, Perez-Pariente J,
Vallet-Regi M. 2004. Micropor mesopor mater 68:105-109.
Example 7. Agarose Surface Coating Influences Intracellular
Accumulation and Enhances Payload Stability of a Nano-Delivery
System
[0282] Protein therapeutics often requires repeated administrations
of the drug over a long period of time. Proteins' instability is a
major obstacle to the development of systems for their controlled
and sustained release. In this work we describe a surface
modification of nanoporous silicon particles (NSP) with an agarose
hydrogel matrix that enhances their ability to load and release
proteins, influencing intracellular delivery and preserving
molecular stability.
[0283] We developed and characterized an agarose surface
modification of NSP. Stability of the released protein after
enzymatic treatment of loaded particles was evaluated with SDS-page
and HPLC analysis. FITC-conjugated BSA was chosen as probe protein
and intracellular delivery evaluated by fluorescence
microscopy.
[0284] We showed that agarose coating does not affect NPS protein
release rate while fewer digestion products were found in the
released solution after all the enzymatic treatments. Confocal
images show that the hydrogel coating improves intracellular
delivery, specifically within the nucleus, without affecting the
internalization process.
[0285] This modification of porous silicon adds to its tunability,
biocompatibility and biodegradability, the ability to preserve
protein integrity during delivery without affecting release rates
and internalization dynamics. Moreover it may allow the silicon
particles to function as protein carriers that enable control of
cell function.
[0286] During the last few decades protein therapeutics has
developed dramatically and gained a significant role in many fields
of medicine (1). Proteins such as growth factors, hormones, and
cytokines are achieving widespread recognition as therapeutic
agents (2), while protein epitopes are now being mapped and used
for vaccination that provides broad protection against infectious
agents (3). Various therapeutic proteins have been proposed in the
literature with a wide range of roles and functions in the body
(4-7): formation of receptor domains on the cell surface,
improvement of the intracellular and/or extracellular molecular
transport, enzymatic catalysis of biochemical reactions, enzymatic
or regulatory activity, targeting, vaccines (8, 9) and diagnostics
(10-12). Protein drugs are able to act selectively on biological
pathways but often require repeated administration, making their
clinical use even more challenging than that of conventional drugs
(13-16). The controlled and sustained release of proteins may
enhance their therapeutic efficacy and reduce the pain and
inconvenience of frequent injections. However, this route of
administration faces a single major issue: protein instability
(17). Proteins are unstable molecules and once injected in the
bloodstream they are rapidly degraded and deactivated by specific
enzymes (18). Growth factors such as FGF and VEGF, for example,
have half-life as short as 3 and 50 minutes respectively (19, 20).
Furthermore sustained release (days to months) and formulation of
the delivery system often exposes the protein to harmful conditions
that disrupt its integrity and ultimately compromises its
therapeutic efficacy (21, 22).
[0287] In the past years, many drug-delivery systems have been
developed. Some organic ones (e.g liposomes, micelles,
nanoparticles) are able to deliver drugs to a specific site and at
the desired rate; yet, most of these systems are rapidly eliminated
by the reticulum endothelial system (RES). Furthermore, polymeric
formulations (such as PLGA), release acidic byproducts upon
degradation, and can induce local inflammatory responses that
negatively impact protein integrity and activity (23, 24).
[0288] Porous silicon (pSi) has been proposed as an ideal
biomaterial for drug delivery thanks to its biocompatibility (25,
26), tunability of the porous structure (27, 28), ease and
versatility of processing through standard semiconductor technology
(29, 30), and for the well-established protocols for the
optimization of its surface chemistry (31, 32). As a result, pSi
has been successfully used to improve drug solubility, increase
bioavailability, and modulate release rates, thus paving a
promising path for the realization of pSi drug delivery devices
(33-35). pSi has been successfully employed for the loading and
release of peptides, proteins and nanoparticles in a controlled and
sustained fashion (35-38). Peptides loaded into porous silicon
particles have been systemically delivered in vivo resulting in a
prolonged effect compared to their free administration (39). Post
synthesis modification of pSi provided controlled release and
enhanced loading of bioactive molecules (33, 36, 37, 40). However,
the stability of the loaded/encapsulated protein has not been
guaranteed thus far.
[0289] This work describes a novel surface modification with
agarose hydrogel developed to enhance protein stability within
nanoporous silicon particles (NSP) during sustained and controlled
release, and during enzymatic digestion. Moreover we report the
coating's control over NSP intracellular trafficking and uptake.
The enhancements to protein delivery of this NSP surface matrix
coating may extend the use of pSi as a versatile delivery system
for enzymes, vaccine antigens, and protein therapeutics in
general.
[0290] Nanoporous Silicon Particles Synthesis and APTES
Modification
[0291] NSP were designed and fabricated in the Microelectronics
Research Center at The University of Texas at Austin by established
methods (29, 35). In brief, after low pressure chemical vapor
deposition of 100 nm silicon nitride (SiN), photoresist was spun
cast on a 100 mm, 0.005 .OMEGA.-cm p-type Si wafer. A pattern
consisting of 2 .mu.m dark field circles with 2 m pitch was
transferred to the photoresist by contact photolitography. Then the
pattern was transferred for 100 nm into the silicon substrate by
reactive ion etching with CF.sub.4 gas. The photoresist was removed
from the substrate for anodic etch preparation by piranha clean.
The porous particles were formed by selective porosification
through the SiN mask by anodic etch. The SiN layer was removed by
soaking in HF, the substrate was dried and the particles were
released in isopropanol by sonication. Particles were then oxidized
by piranha (solution of 2:1 vol. H2SO4 (96%) in H2O2 (30%)) for 2 h
at 120.degree. C. Then modified with aminopropyltriethoxysilane
(APTES--2% in IPA) for 2 hours at 35.degree. C. to provide a
controlled positive charge to the particle surface that enhances
protein loading capacity.
[0292] Modification of Nanoporous Silicon Particles with Agarose
Matrix
[0293] Agarose coating was performed by suspending NSP in warm
(40.degree. C.) agarose solution for 15 minutes and then the
solution was cooled at 4.degree. C. for 30 min. Agarose coating
solutions were prepared at different concentrations ranging from
0.05 to 0.5%.sub.w with low melt certified agarose (BIORAD), used
as received. To remove excess gel, particles were washed with warm
PBS (35.degree. C.) and cooled at room temperature twice. Agarose
coating of loaded NSP was performed after loading before any
washing step.
[0294] NSP Characterization
[0295] The volume, size and concentration NSPs were characterized
by a Multisizer.TM. 4 Coulter Counter (Beckman Coulter). Before the
analysis, the samples were dispersed in the balanced electrolyte
solution (ISOTON VR II Diluent, Beckman Coulter Fullerton, Calif.)
and sonicated for 5 s to ensure a homogenous dispersion. Their
surface charge before and after APTES modification and agarose
coating was measured in a PB buffer at pH 7.4 using a ZetaPALS Zeta
Potential Analyzer (Brookhaven Instruments Corporation; Holtsville,
N.Y.). The surface area and pore size distribution of the NSPs were
measured using N2 adsorption-desorption isotherms on a Quantachrome
Autosorb-3B Surface Analyzer. To prepare the sample, 10 mg of NSPs
was transferred to a sample cell, and dried in a vacuum oven at
80.degree. C.
[0296] The sample was degassed at 200.degree. C. for 12 hours, and
the N2 adsorption-desorption isotherm was measured at 77K over the
relative pressure (P/PO) range of 0.015-0.995. Nanopore size
distributions and porosities were calculated from the desorption
branch of the isotherms using the BJH model. NSP size and shape was
also evaluated at different timepoints during incubation in PBS at
room temperature by scanning electron microscope (SEM) (FEI Quanta
400 ESEM FEG). To prepare SEM sample, a drop of PSN IPA solution is
directly placed on a clean aluminum SEM sample stub and dried. Ag
samples were sputter-coated with gold for 2 min at 10 nm layer
using a CrC-150 Sputtering System (Torr International, New Windsor,
N.Y.). All the samples were loaded in SEM chamber, and SEM images
were measured at 5 kV and 3-5 mm working distance using an In-lens
detector. Size variation over time was also examined by
fluorescence activated cell sorting (FACS) (Becton Dickinson,
FACSCalibur). Solution pH was measured with pH strips
(colorPHast--EMD).
[0297] Protein Loading and Release
[0298] Lyophilized and fluorescein isothiocyanate (FITC) conjugated
bovine serum albumin (BSA) was chosen as a protein probe, purchased
from Sigma-Aldrich, and used as received. BSA was loaded into NSP
by suspending 10.sup.8 NSP in 200 .mu.L of 25 mg/mL BSA (1.2% of
BSA was FITC-conjugated) aqueous solution (prepared in PBS--GIBCO
Invitrogen). The suspension was continuously mixed in dark at
4.degree. C. for 2 hours, then spun down and the supernatant was
removed. To remove excess of probe three washing steps were
performed. Coated and not coated particles underwent the same
number of washing steps.
[0299] To measure the loading efficiency of NSP, the fluorescence
and concentration of the BSA solution used for the loading (as
prepared for the loading procedure and as recovered after
incubation), was quantified by spectrofluorimetry with SpectraMax
M2 spectrophotometer (Molecular Devices). The BSA loss during
coating procedure was also taken into account by measuring coating
and washing solutions fluorescence/concentration.
[0300] Protein release over time from NSP (bare (not coated--NC)
and agarose coated (Ag) with two agarose concentrations (0.05 and
0.125%.sub.w)), was studied by collecting all the supernatants and
replacing them with fresh PBS at each timepoint. Release
quantification was performed measuring protein content in the
supernatant with the Bradford method, by spectrofluorimetry and by
FACS (Becton Dickinson, FACSCalibur).
[0301] Protein Stability Analysis
[0302] NC and Ag (0.125%) NSP loaded with BSA, were treated with
trypsin (25 .mu.g/mL) for different times and enzymatic digestion
was ended adding equal volume of bleaching solution (20%
acetonitrile-CH.sub.3CN and 4% trifluoroacetic acid-TFA in water)
at the different time points. The structural integrity of the BSA,
released after 24 hours from NC and Ag NSP after the different
trypsin treatments, was analyzed with sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-page) using Criterion
Tris-HCl Gel (BioRad) in non-reduced condition and high performance
liquid chromatography (HPLC) (ELITE LaChrome, Itachi). Digestion
products were also quantified analyzing SDS page silver stained
bars with ImageJ.
[0303] Cell Culture and Confocal Microscopy
[0304] Human umbilical vein endothelial cells (HUVEC) were cultured
in complete Dulbecco's modified eagle's medium (DMEM) at 37.degree.
C. and in 5% CO.sub.2 using 2 different systems: (a) 4 chamber
tissue culture treated glass slides and (b) circular glass
coverslip of 8 mm diameter placed in 12 well plates. 120,000 and
240,000 cells were seeded per chamber and well respectively. Cells
were allowed to settle for 2 hours before adding NSP. On the glass
slide 600,000 NC or Ag NSP loaded with BSA FITC-conjugated were
added directly to the cells in each chamber and incubated for 24
and 48 hours. In the multi-well plate 1,200,000 NC or Ag NSP loaded
with BSA FITC-conjugated were added in a transwell over the cells
to each well, avoiding direct contact between cells and NSP.
[0305] Cellular internalization of NSP and uptake of BSA were
observed for both systems by confocal microscopy (Leica MD 6000)
after 24 and 48 hours incubation with Ag or NC particles. Cells
were stained with fluorescent phalloidin (actin filaments) and
DRAQ5 (nuclei) after fixation in 4% paraformaldehyde. Cellular
uptake of BSA from 1 mg/mL BSA-FITC conjugated solution prepared in
DMEM was also evaluated. All images used for quantification were
acquired by keeping the same acquisition setting (pinhole, gain,
laser power, optical path, line average, zoom and image resolution)
for the whole duration of the experiment. Numerical evaluation of
the fluorescence was performed using the Nikon Elements software.
The average fluorescence within the cytoplasm or the nuclei was
measured in different, representative field of views (at least 5
cells per image per timepoint). Cellular uptake of BSA from protein
dispersed in solution was not numerically quantified because by
using the same confocal setting most of the cells appeared
supersaturated thus not allowing a direct comparison between the
two conditions.
[0306] Statistical Analysis
[0307] Reported data are the averages of at least three different
measurements, and statistical significance (p<0.05) was
evaluated with ANOVA (Origin), if not otherwise stated in the
text.
[0308] Characterization of Nanoporous Silicon Particles
[0309] NSP used in this work are quasi-hemispherical shells of 3.2
.mu.m diameter and 600 nm shell thickness (FIG. 35A and FIG. 35F)
designed for drug delivery application (41). Pore size is 15 nm
with 51% porosity as estimated from the desorption branch of
nitrogen adsorption/desorption isotherms. APTES modification
altered particles' surface charge (zeta potential from -23 mV to +1
mV) and allowed the loading of about 10 .mu.g of BSA per million of
NSP (7 .mu.g). BSA is negatively charged and could not be loaded in
oxidized particles (loaded particle zeta potential was -28 mV).
[0310] The agarose coating was developed and optimized to assure a
protective function against harmful agents during long-term
release. SEM images (FIGS. 35B-35E) indicated that the resulting
agarose coating was uniform and density increased with agarose
concentration. Agarose hydrogel matrix filled the pores and covered
the particles' surface completely but did not alter appreciably the
size and charge of the NSP (zeta potential was +2 and -30 mV for
not loaded and loaded NSP respectively).
[0311] Agarose coatings appeared to be uniform and smooth for all
conditions considered. At the highest agarose concentrations (0.25
and 0.5%) hydrogel residues and particle aggregates appeared (see
supplementary information). To assure stable uniform coating and
good dispersion of the particles, 0.05 and 0.125% agarose
concentrations (A1 and A2 respectively) were selected for further
analysis, together with bare (not coated--NC) NSP for
comparison.
[0312] Degradation process of NC NSP as observed at SEM is shown in
FIGS. 36A-36H.
[0313] SEM images show the progressive degradation of NSP (into
orthosilicic acid as assessed by ICP, data not shown (35, 42, 43))
during degradation while their size slightly decreased. Degradation
rate of exposed silicon was uniform across the entire particle. As
previously reported, we observed higher degradation in the outer
rim because of the higher surface area and porosity of this
structure (42).
[0314] NSP degradation over time was also monitored with flow
cytometry (FACS) (FIG. 37A-37N) quantifying NPS size variation
through the change in the forward scattering intensity. Polystyrene
beads of given size were used as calibration standards. FACS data
showed that NPS size reduced in three days from about 3 to almost 2
Dm (FIGS. 37A, 37D, 37H, and 37K) as was observed also at the SEM.
FACS analysis reveals no significant differences between NC and Ag
NSP with either agarose concentrations (A1 and A2) (FIGS. 37A-37N
and FIGS. 370-37P)..
[0315] Quantification of Protein Release
[0316] To assess protein release from Ag NSP, fluorescent BSA was
used as model. Loading and release of BSA from Ag NSP with two
agarose concentrations (A1 and A2) and NC NSP were quantified by
fluorescence spectroscopy. Loading efficiency was about 70% for
both NC and Ag NSP (FIG. 38A); hence the agarose coating did not
affect protein loading. FACS and spectrometric BSA release data are
shown in FIGS. 38B and 38C respectively.
[0317] FACS results (FIG. 38B) showed that NSP fluorescence
exponentially decreased (y=A*e .sup.B*x-R.sup.2>0.91) in three
days. Moreover there was no significant difference between NSP NC
and Ag with both agarose concentrations. Spectrofluorimetry data
(FIG. 38C) also showed that all the loaded protein was released
with a logarithmic profile (y=A*ln(x)+B-R.sup.2>0.98) within
three days for NSP NC and Ag with both agarose concentrations. FACS
and spectrofluorimetry data agreed showing that while the BSA was
released from NSP the particles' fluorescence decreased accordingly
(see supplementary information for fitting curves and parameters);
after 3 days almost all BSA was released (.about.90%) and NSP were
almost not fluorescent anymore (.about.5%). Protein release study
results indicated that agarose coating does not affect protein
release from NSP.
[0318] Released Protein Integrity Analysis
[0319] To assess the protection of protein integrity provided by
agarose coating, BSA loaded NSP were treated with trypsin for 10,
30, 60 and 120 minutes, and released BSA solution analyzed with SDS
page. Resulting gel for NC and Ag (composition A2) NSP is shown in
FIG. 39A.
[0320] The gel analysis showed several protein fragments, digestion
products, together with BSA and trypsin (when added), and no
aggregates (see supplementary information). The concentration and
number of fragments appeared higher in the solutions released from
the NC NSP. Moreover the presence of protein fragments increased
with trypsin treatment time while trypsin and BSA amounts were
about the same in all the samples.
[0321] To better quantify protein, enzyme, and digestion products
the SDS result was also analyzed with ImageJ and the three most
abundant digestion products plotted as function of trypsin
treatment duration (FIGS. 39B-39D). The quantitative analysis
showed that solution recovered from NC NSP samples contained a
higher concentration of digestion products than the one recovered
from Ag NSP for all treatment conditions. The samples not treated
with trypsin showed no difference between NC and Ag NSP. The amount
of BSA and trypsin was the same in all treated samples. The amount
of fragments increased with trypsin treatment time for the NC NSP
samples but was almost constant in the Ag NSP ones.
[0322] HPLC analysis performed on BSA solution recovered after 24
hours from NSP not treated and treated with trypsin for 15 minutes,
2 hours, 4 hours, 8 hours and 18 hours is shown in FIGS.
40A-40B.
[0323] Graphs show an increase of digestion products concentration
and number with duration of trypsin treatment. There were more
digestion products in the solution released by NC particles
especially for longer trypsin treatment time, as evidenced
especially for the three species pointed by the arrows. These
results are in agreement with the SDS-page analysis and confirm the
protective function of the agarose coating from enzymatic
digestion.
[0324] Cellular Internalization of NSP and Uptake of Protein
[0325] Cellular uptake of protein was studied using fluorescent BSA
and evaluating the fluorescence within HUVEC by confocal microscope
imaging after 24 and 48 hours. Particles internalization and BSA
uptake after 48 hours of incubation with NC and Ag (composition A2)
NSP added into the media with the cells or in a transwell on top of
them is shown in FIGS. 41A-41F.
[0326] After 48 hours of incubation with cells, both NC and Ag NSP
were completely internalized and BSA was released within the cells.
Confocal microscopy showed that the internalization process was not
affected by the agarose coating and NSP accumulated in the
lysosomes in less than 1 hour, as previously reported (44).
[0327] NSP internalization was inhibited using the transwells and
BSA was first released in the media and then incorporated into the
cells. Images show that uptake of BSA released from NSP in the
transwell or from BSA solution was not uniform within the cells and
the protein probably accumulated within the lysosomes. The
fluorescence within the cells receiving BSA from the transwell was
comparable with that of the cells that internalized NSP. The
cellular uptake of BSA from protein dispersed in solution (1 mg/mL)
appears higher than the one achieved by NSP release (to avoid pixel
oversaturation, different confocal settings were used to acquire
FIG. 41F).
[0328] This can be attributed to less BSA being released from NPS
resulting in a lower overall BSA concentration in the media. A
difference in the cellular uptake of BSA between internalized Ag
and not coated particles was observed. We hypothesized that the
agarose coating was able to induce a change of pH within the
lysosomes and influence the cellular uptake. To assess if the
agarose coating matrix would affect the pH within the lysosomes,
different volumes of pH 5 solution and agarose coating solution
were mixed at room temperature and the change of pH was measured
(FIG. 42).
[0329] As shown in FIG. 42, pH increased from 5 to 6 or more,
depending on the ratio of agarose coating solution (AG), while no
change of pH was observed if agarose was prepared with DI water
instead of PBS. This experiment revealed that the agarose solution
used to coat the particles had a buffering capacity which could
have been instrumental for the local modification of the pH in the
small acidic lysosomal environments.
[0330] The progression over time of the uptake process relative to
HUVEC incubated with NC and Ag NSP is shown in FIGS. 43A-43D. After
24 hours of NSP incubation, cellular uptake of BSA was visible but
still not evident especially for NC NSP. BSA accumulated in the
cells where the NSP, both NC and Ag particles, were internalized.
The protein, escaping from the lysosomes, was uniformly distributed
throughout the nuclei and the cytoplasm of the cells. We
hypothesize that the agarose coating affected lysosome pH once NSP
were internalized and hence facilitated protein escape.
[0331] To better quantify the BSA uptake within the cells, the
average green fluorescence intensity of confocal images within the
cytoplasm and the nucleus of the cells was quantified with Elements
(Nikon) and correlated with the number of NSP internalized in each
cell (FIGS. 43E-43F). Data showed a higher uptake of BSA within
cells incubated with Ag NSP than with NC NSP. Uptake of the protein
was also proportional to the number of particles internalized.
Uptake of BSA released from Ag NSP increased more rapidly with the
number of internalized NSP than from NC NSP. Additionally protein
accumulated within the nuclei more than within cytoplasm.
[0332] These data suggested that agarose coating increases cellular
uptake of the protein and avoids extended entrapment in the
lysosomes.
Conclusion
[0333] In this work we successfully modified with hydrogel NSP,
designed and fabricated for drug delivery application, to improve
their efficacy for intracellular protein release. We verified that
the agarose coating protects the payload from enzymatic digestion
while it does not affect its release from the NSP. We also showed
that the hydrogel coating increases cellular uptake and influences
intracellular trafficking of the protein in comparison with what
was observed from proteins dispersed in solution. Furthermore the
agarose coating is able to improve intracellular protein delivery
and increases the accumulation of the protein within the nuclei.
Thus the agarose coating of NSP may extend the use of pSi as
versatile delivery system for enzymes, vaccine antigens, gene
therapy and other protein therapeutics. Additionally it may act
effectively in combination with other controlled release systems
(e.g. PLGA encapsulation) to preserve protein stability during
controlled drug delivery formulation and long term release.
[0334] Notations
[0335] FGF=fibroblast growth factor, VEGF=vascular endothelial
growth factor, BSA=bovine serum albumin,
PLGA=poly(lactic-co-glycolic acid), NSP=nanoporous silicon
particles, NC=bare-not coated, Ag=agarose coated, A1=agarose
composition 0.125%, A2=agarose composition 0.05%,
APTES=aminopropyltriethoxysilane, pSi=porous silicon, SiN=low
stress silicon nitride, SEM=scanning electron microscope,
FACS=fluorescence activated cell sorting, SDS-page=sodium dodecyl
sulfate polyacrylamide gel electrophoresis, HPLC=high performance
liquid chromatography, HUVEC=human umbilical vein endothelial
cells.
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porous silicon powders. physica status solidi (a). 204:3361-3366
(2007). [0373] 38. R. E. Serda, B. Godin, E. Blanco, C. Chiappini,
and M. Ferrari. Multi-stage delivery nano-particle systems for
therapeutic applications. Biochimica et Biophysica Acta
(BBA)--General Subjects. In Press, Corrected Proof:in press (2010).
[0374] 39. M. Kilpeliinen, J. Riikonen, M. A. Vlasova, A. Huotari,
V. P. Lehto, J. Salonen, K. H. Herzig, and K. Jiirvinen. In vivo
delivery of a peptide, ghrelin antagonist, with mesoporous silicon
microparticles. Journal of Controlled Release. 137:166-170 (2009).
[0375] 40. E. J. Anglin, M. P. Schwartz, V. P. Ng, L. A. Perelman,
and M. J. Sailor. Engineering the Chemistry and Nanostructure of
Porous Silicon Fabry-Perot Films for Loading and Release of a
Steroid. Langmuir. 20:11264-11269 (2004). [0376] 41. M. Ferrari.
Nanogeometry: Beyond drug delivery. Nat Nano. 3:131-132 (2008).
[0377] 42. B. Godin, J. Gu, R. E. Serda, R. Bhavane, E. Tasciotti,
C. Chiappini, X. Liu, T. Tanaka, P. Decuzzi, and M. Ferrari.
Tailoring the degradation kinetics of mesoporous silicon structures
through PEGylation. Journal of Biomedical Materials Research Part
A. 94A: 1236-1243 (2010). [0378] 43. R. E. Serda, A. Mack, M.
Pulikkathara, A. M. Zaske, C. Chiappini, J. R. Fakhoury, D. Webb,
B. Godin, J. L. Conyers, X. W. Liu, J. A. Bankson, and M. Ferrari.
Cellular Association and Assembly of a Multistage Delivery System.
Small. 6:1329-1340 (2010). [0379] 44. S. Ferrati, A. Mack, C.
Chiappini, X. Liu, A. J. Bean, M. Ferrari, and R. E. Serda.
Intracellular trafficking of silicon particles and logic-embedded
vectors. Nanoscale. 2:1512-1520 (2010).
Example 8. Mesoporous Silicon-PLGA Composite Microspheres for the
Double Controlled Release of Biomolecules for Orthopedic Tissue
Engineering
[0380] In this study, PLGA/pSi composite microspheres, synthesized
by a solid-in-oil-in-water (S/O/W) emulsion method, are developed
for the long-term controlled delivery of biomolecules for
orthopedic tissue engineering applications. Confocal and
fluorescent microscopy, together with material analysis show that
each composite microsphere contained multiple pSi particles
embedded in the PLGA matrix. The release profiles of FITC
labeled-Bovine Serum Albumin (FITC-BSA), loaded in the pSi within
the PLGA matrix, indicate that both PLGA and pSi contribute to
control the release rate of the payload. Protein stability studies
show that PLGA/pSi composite can protect BSA from degradation
during the long term release. We find that during the degradation
of the composite material, the presence of the pSi particles
neutralizes the acidic pH due to the PLGA degradation by-products,
thus minimizing the risk of inducing inflammatory responses in the
exposed cells while stimulating the mineralization in osteogenic
growth media. Confocal studies show that the cellular uptake of the
composite microspheres is avoided, while the fluorescent payload is
detectable intracellularly after 7 days of co-incubation. In
conclusion, the PLGA/pSi composite microspheres could be ideal
candidates as drug delivery vehicles for orthopedic tissue
engineering applications.
[0381] Introduction
[0382] Porous silicon (pSi) has been widely used for tissue
engineering and drug delivery in virtue of its biodegradable and
biocompatabile nature..sup.[1] As a scaffold, pSi is suitable for
directing the growth of neuronal cells.sup.[2] and for stimulating
mineralization in bone tissue engineering..sup.[3,4] For
therapeutic delivery, pSi has been administered orally,.sup.[5]
intravenously,.sup.[6] or been injected percutaneously and
intraperitonealy in humans for brachytherapy without notable side
effects..sup.[7] A wide variety of therapeutic and imaging agents
have been successfully loaded into and released from pSi particles
including steroids,.sup.[8] hormones,.sup.[9] proteins,.sup.[10]
cancer drugs,.sup.[11] iron oxide nanoparticles,.sup.[12] quantum
dots, liposomes.sup.[13] and carbon nanotubes.sup.[14,15] showing
the great versatility of this material as a delivery system. Also,
the size and shape as well as the porosity and pore size of the pSi
particles can be engineered and tightly controlled during
manufacturing in order to provide a material with constant and
uniform physical features at the micro- and nano-scale and to
control degradation time and kinetics as well as biodistribution
and bioaccumulation..sup.[16] Additionally, their surface can be
functionalized to accommodate various drugs, control cellular
uptake, target specific tissues.sup.[17] and alter their
biodistribution in murine models,.sup.[13,18] thus allowing for the
accumulation of therapeutic agents at tumor sites,.sup.[19] or in
reservoirs able to sustain the release of nanoliposomes carrying
siRNA..sup.[20]
[0383] Also PLGA, an FDA approved biodegradable polymer, has been
widely investigated for drug delivery applications due to a number
of advantageous features..sup.[21,22] First, its degradation rates
can be tailored to obtain controlled delivery of drugs. Secondly,
the material properties can be adjusted by changing the lactic acid
and glycolic acid ratio or molecular weight. Thirdly, PLGA
nanoparticles or microparticles can be formulated in order to load
not only small molecules but also proteins and larger
payloads..sup.[23,25] However, some issues that remain unsolved
include the achievement of a uniform, zero order, sustained, linear
release and to prevent the initial burst release typical of most
PLGA systems..sup.[26] Additionally, the acidic PLGA degradation
by-products decrease the pH of the surrounding environment, which
may cause undesired inflammatory responses..sup.[27] Finally, the
available fabrication methods for PLGA microparticles are
incompatible with water-soluble proteins as they may degrade or
denature at the organic/inorganic interface during formulation
processes..sup.[28]
[0384] In this study we show that the addition of pSi particles to
PLGA microspheres offers a solution to each one of the
aforementioned issues. pSi particles due to their high surface area
and to their interconnected pores allow for the storage and
protection of large amounts of therapeutic molecules..sup.[29]
Additionally, PLGA coating provides a tunable layer to seal pSi
pores, slow down pSi degradation, and control the release of the
payload. Orthosilicic acid, the by-product of pSi
degradation,.sup.[30] can neutralize the acidic pH of the PLGA
degradation products thus creating less harsh and more cell
friendly conditions in the microenvironment both in vitro and in
vivo..sup.[31] The use of hydrophilic pSi particles increased the
hydrophilicity of the PLGA/pSi system and improved cell anchorage
while not affecting cell proliferation. When the soluble proteins
were efficiently loaded within the pores of the pSi particles,
their structural integrity (biostability) was preserved.
Furthermore, orthosilicic acid is involved in the collagen
formation and facilitates the deposition of calcium and other
minerals, thus stimulating bone formation in orthopedic tissue
engineering applications..sup.[32,33]
[0385] pSi Particles
[0386] Quasi-hemispherical shaped pSi shells of 3.2 .mu.m in
diameter and 600 nm shell thickness (as shown in FIGS. 45A-45D)
were fabricated according to established protocols..sup.[14]
Average Pore size was 20 nm with 51% porosity as determined from
the desorption branch of nitrogen adsorption/desorption isotherms
(data shown in supporting material). In order to turn the pSi
surface from hydrophobic to hydrophilic, the pSi surface was
modified with (3-Aminopropyl) triethoxysilane (APTES). Zeta
potential analysis showed that the surface charge of the particles
after APTES modification had a value of 6.44 mV, while the oxidized
pSi surface had a surface charge of -30.39 mV. Once resuspended in
IPA, the surface charge of the APTES modified particles showed no
notable change for 2 weeks, thus indicating stable modification of
the exposed silicon layer (data shown in FIGS. 56-58).
[0387] PLGA/pSi Microsphere Characterization
[0388] The overall aspect and the morphology of the microspheres
were characterized by optical, confocal, and scanning electron
microscopy. FIG. 46A shows the SEM images of FITC-BSA loaded
microspheres. FIG. 46B shows the transmission microscopy image of
the composite material that allows to appreciate the pSi particles
(brown dots, see arrows) embedded in the transparent spherical PLGA
particles. These images indicate that the pSi particles had been
fully encapsulated in the PLGA spheres. Fluorescent microscopy
image (FIG. 46C) shows the same results. FITC-BSA diffused from pSi
particles into the PLGA layer. FIG. 46D shows the size distribution
of the PLGA/pSi microspheres. The microspheres displayed a
distribution of sizes ranging from a few microns to approximately
35 .mu.m with an average diameter of 24.5.+-.9.54 .mu.m (145
microspheres were measured).
[0389] PLGA/pSi Microsphere Sorting
[0390] PLGA/pSi microspheres prepared with 488-DyLight conjugated
pSi particles were characterized before and after centrifugation
sorting by fluorescence activated cell sorting (FACS) and confocal
microscopy (FIG. 47). FACS data (FIGS. 47A-47B) show that the mean
fluorescence and hence the percentage of coated particles increases
of about one order of magnitude after centrifugation sorting. The
coated-fluorescent fraction was initially only the 10% of the
sample and after the centrifugation process, it increased to 80%.
Moreover the negligible fluorescence of the supernatant reveals the
absence of coated particles in it. Its mean fluorescence is two
order of magnitude lower than the original sample and only 1% of it
has a comparable fluorescence (FIGS. 47A-47B). FIG. 47C shows the
fluorescence intensity and distribution of 488-DyLight conjugated
pSi particles (light green), nonsorted microspheres (blue), sorted
microspheres (dark green), and supernatant solution (black).
[0391] Confocal images show a mix of fluorescent and not
fluorescent microspheres with polydistributed sizes in the not
sorted sample (FIG. 47D) and a more uniform particles size after
the sorting procedure (FIG. 47E) confirming that the sequential
centrifugation procedure achieved a good separation of PLGA/pSi
microspheres from smaller empty PLGA microspheres.
[0392] Evaluation of FITC-BSA Loading
[0393] The mechanism of loading and retention of the molecules
inside the pores of the pSi particles is based on the electrostatic
interactions between the amino groups on the surface of the APTES
modified pSi particles and the carboxylic portion of the amide
groups in the protein. The loading efficiency of FITC-BSA into pSi
particles varied between 9.77% to 86%, depending on the
concentrations of the loading solutions (data shown in supporting
material). During the microemulsion step, the loss of the FITC-BSA
was approximately 13.24%, 9.84%, and 5.14% from the composites and
it inversely correlated with the density of the different PLGA
coatings (6%, 10%, and 20% respectively). This result demonstrated
that during synthesis higher concentrations of the coating
solutions resulted in lower protein loss.
[0394] In Vitro Release of FITC-BSA
[0395] The release profiles of FITC-BSA from pSi particles
(control), PLGA microspheres (control) and PLGA/pSi microspheres
are shown in FIG. 48A. In the case of pSi particles and PLGA
microspheres (6%, 10%, and 20%), protein release showed a massive
initial burst release which reached the plateau after less than 3
days. On the contrary, PLGA/pSi microspheres released approximately
70% (6% PLGA), 38% (10% PLGA), and 25% (20% PLGA) of the payload at
day 3 (FIG. 48B). After 2 weeks, the release of FITC-BSA from the
composite reached approximately 100% (6% PLGA/pSi), 60% (10%
PLGA/pSi), and 40% (20% PLGA/pSi) of the payload as shown in FIG.
48C and it continued to be released for other 2 weeks from the
higher density PLGA coatings (10% and 20%) (FIG. 48D). FIGS. 49A
and 49B show that at all time points, PLGA/pSi microparticles
showed consistently higher fluorescence when compared to controls.
Due to the initial burst release of FITC-BSA during the first 3
days, the fluorescence of PLGA microspheres decreased at fast pace
and dropped to its minimum. Conversely, the addition of pSi
particles to the PLGA microspheres reduced the FITC-BSA release
rate as demonstrated by the higher fluorescence intensity measured
throughout the experiment.
[0396] Altogether, the study of the in vitro release of FITC-BSA
demonstrated that the PLGA coating played an important role in
controlling the release kinetics from the microspheres. A higher
concentration of PLGA resulted in a coating layer characterized by
higher density and thickness. As a consequence, the diffusion of
FITC-BSA through the PLGA layers was slowed down, resulting in
lower release rates and more sustained delivery of the payload.
Similarly, a thicker layer of PLGA delayed the degradation of the
composite microspheres thus additionally slowing down the release
of the encapsulated proteins. In all profiles, the two phases
observed during protein release were attributed to a minor fraction
of the pSi loaded BSA which diffused into the PLGA layer during the
microsphere fabrication process and was released earlier than the
fraction still loaded into the pores of pSi particles.
[0397] PLGA/pSi Microsphere Degradation
[0398] The PLGA/pSi microsphere degradation was studied by
monitoring the morphology changes using SEM. FIGS. 50A-50H and
FIGS. 50J-50P show the SEM images of three types of PLGA/pSi
microspheres (6%, 10%, and 20% PLGA coatings) degradation over 6
weeks in PBS. At week 1, pores were observed on the surface of all
three types of microspheres, showing an early-stage degradation.
Pore number and size increased with time and after 3 weeks, 6% and
10% PLGA/pSi microspheres appeared deformed and partially
collapsed. At week 4, more pores appeared on the surface of the 6%
PLGA/pSi microspheres, while the surface layers of polymer coatings
were peeled off from the 10% and 20% PLGA/pSi mircospheres and a
porous, sponge-like morphology was observed beneath the surface. At
week 6, the 6% PLGA/pSi microspheres completely lost their
spherical morphology, while the 10% and 20% PLGA/pSi broke into
pieces revealing the inner porous structure of the microsphere.
[0399] FIGS. 51A-51B demonstrate the change of pH in the medium
during the degradation of PLGA, pSi, and PLGA/pSi microspheres. The
control sample (pSi) kept a constant pH value of approximately 7.2
during the 4-week degradation. PLGA microsphere degradation induced
a pH drop at two weeks (FIG. 51A). However, when pSi microparticles
were introduced to the PLGA microspheres, the pH values recorded
were approximately around 7 over the four-week degradation period,
and only the microspheres with the thickest coating (20% PLGA)
generated acidic conditions after four weeks (FIG. 51B). This is
due to the fact that the pSi degradation product, silicic acid
buffered the pH at higher values..sup.[34,35] The mass ratio of
PLGA to pSi is 5:1 (6% PLGA/pSi), 8:1 (10% PLGA/pSi), and 16:1 (20%
PLGA/pSi). The PLGA and pSi particles degrade concurrently, which
allows silicic acid to buffer the acidic environment when the
acidic products of PLGA are produced. As expected, lower ratios
showed higher buffer capacity than the higher ratio.
[0400] BSA Stability Studies
[0401] BSA, like all other proteins, is susceptible to hydrolytic
degradation in aqueous solutions. These reactions can be catalyzed
by acidic molecules, such as the byproducts of PLGA. In order to
minimize protein degradation during loading, FITC-BSA was first
loaded into pSi microparticles and lyophilized prior to PLGA
coating. This step reduces exposure to water during particle
preparation and during eventual PLGA degradation. SDS-PAGE of
released FITC-BSA and degraded byproducts is exhibited in FIG. 52.
The appearance of bands for degraded proteins is substantially less
for PLGA/pSi-released BSA than controls at 7 days. Between 9 and 14
days, a relatively small amount FITC-BSA is released which is
insignificant compared to controls and 7 day time points. However,
10% and 20% coating groups show only an intact FITC-BSA band and
not small byproducts, indicating that molecules released after one
week have not been hydrolytically degraded. This is likely because
molecules stored deep within the core of the microparticles are not
exposed to any water until the PLGA coating has been sufficiently
eroded.
[0402] In Vitro Mineralization Studies
[0403] PLGA microspheres do not calcify in the absence of bioactive
materials which stimulate deposition of calcium phosphate (CaP)
bone mineral. This study has investigated if the addition of pSi
microparticles to PLGA microspheres can render these inert
microspheres bioactive. After incubation in the osteogenic media
for 3 days, the smooth surface of the PLGA/pSi microspheres was
covered with a porous rough layer (FIGS. 53A-53G), while the
control PLGA microspheres remained smooth with just minimal crystal
deposition on the surface (FIG. 53A). After 21-day incubation, SEM
images showed that the surface of PLGA/pSi microspheres was
uniformly covered with a layer of mineral deposits (FIG. 53D) while
the control samples showed negligible signs of calcification under
the same conditions at the same time intervals (FIG. 53C). This
phenomenon was confirmed at higher magnification at SEM (FIG.
53E-53F). These data suggested that the pSi contained in the PLGA
microspheres has the ability to stimulate the formation of a
mineralized layer on the surface. As a confirmation of the
formation of the calcium phosphate crystals on the surface of the
microspheres, in the EDX spectrum showed calcium and phosphorous
peaks on the surface layer at day 3 (grey dot line) and day 8
(black solid line) (FIG. 53G). The mechanism of calcium phosphate
deposition is that the polymerized silicic acid acted as
heterogeneous nucleation substrate to stabilize the growing of
calcium phosphate nuclei. The uniformly coated osteoactive mineral
layer will further enhance the osteogenic qualities and the
osteoconductive potential of the scaffolds, while still allowing
the release of the bioactive molecules due to the inherent porosity
of the surface mineralization (see FIG. 9F).sup.[33]
[0404] PLGA/pSi Microsphere Internalization by BMSCs
[0405] Most growth factors and differentiating stimuli function by
binding to cell surface receptors to start active transmembrane
signal transduction while the ligand is still in the extracellular
space. When growth factors or differentiation stimuli are vehicled
by a nanosized carrier and the carrier is internalized by the
target cells, they fail to interact with the membrane receptors and
hence, completely lose their function and bioactivity..sup.[36] The
intended function of our composite particles is to release
bioactive proteins at the site of tissue repair. In these
scenarios, macrophages and other inflammatory cells often
internalize and degrade nano-size particles through endocytosis,
pinocytosis and phagocytosis..sup.[37,38] In this study, BSA was
used as a moel growth factor to be delivered by PLGA/pSi
microspheres. The PLGA coating around pSi particles prevents
internalization due to its size, while providing a hydrophobic
barrier to enzymes released by the cells thus protecting for longer
times their bioactive payloads. One of the purposes of this study
was to determine if the PLGA/pSi microspheres could serve as
potential vehicles to successfully deliver growth factors. Confocal
microscopy images showed that the 10% PLGA/pSi microspheres
(average diameter 24.5 .mu.m) were not internalized by the cells
after 0.5 h (FIGS. 54D and 54G), 48 h (FIGS. 54E and 54H) and 120 h
incubation (FIGS. 54F and 54I). The control images showed
accumulation of the uncoated pSi (.about.3 micron) inside the bone
marrow stromal cells within an hour from the beginning of the
incubation (FIG. 54A, 30 min incubation) and after 48 h (FIG. 54B)
and 120 h incubation (FIG. 54C). No cell death, morphological
changes or overall cytotoxicity to BMSCs was observed in vitro
during the entire cell culture period, confirming the compatibility
of these composite microspheres to cells and surrounding
environment. FIG. 54J shows a cartoon describing the mechanism of
action of the pSi particles (right side of the dashed line) versus
the PLGA/pSi composite microspheres (left side of the dashed line).
While pSi are internalized by BMSCs (FIG. 54J), the PLGA/pSi
particles lay on the surface of the BMSCs avoiding cellular uptake
(FIG. 54J).
[0406] Furthermore, the internalization of the pSi inside the cell
would inevitably result in its entrapment into the lysosomal
compartment as shown in FIG. 54J. The acidic environment of
lysosomes would denature the growth factors, affect their
bioactivity and natural site of action thus resulting in the
complete absence of a response to the treatment (FIG.
54J)..sup.[39,40] On the contrary, the ability of the PLGA/pSi
microspheres to escape internalization results in the double
advantage of preventing the exposure of the payload to the hostile
lysosomal environment while releasing it in close contact to the
external layer of the cellular membrane where most of protein
mediated signaling starts. As a consequence of membrane receptor
triggering, the signal pathway arrives to the nucleus thus allowing
for a change in cell functions (color change in FIG. 54J).
[0407] Cellular Uptake of FITC-BSA Released from PLGA/pSi
Microspheres
[0408] BSA, like many growth factors, is internalized through
receptor-mediated endocytosis (clathrin-mediated endocytosis) and
fluid phase endocytosis,.sup.[41-46] and was selected as a model
protein for the release and cellular uptake studies. As mentioned
previously, BSA released from PLGA/pSi microspheres first activated
cell surface receptors to start signal transduction to alter
intracellular response and then BSA was internalized by the cells
(FIG. 54J). To assess the rate of cellular uptake of the BSA
released from the PLGA/pSi microspheres, human umbilical vein
endothelial cells (HUVEC) were studied using confocal microscopy
after 7 days in culture. HUVEC cells were plated in a transwell
without microspheres and incubated with PLGA/pSi in the top chamber
(FIGS. 55A-55E). Confocal images show an evident cellular uptake of
BSA after 7 days of incubation with PLGA/pSi microspheres. Cellular
uptake appeared in discrete spots that probably suggesting protein
accumulation in subcellular organelles. The control group (BSA in
solution) did not show any BSA accumulation in cells. Fluorescence
quantification of the confocal images showed 35 fold increase of
the corresponding green fluorescence, while no difference was
recorded in the red and blue fluorescence associated to the
cytoskeleton (actin) and nucleus (blue) respectively. These results
suggest that PLGA/pSi microspheres can be used as tunable carriers
for releasing bioactive proteins to cells in a controlled and
predictable fashion.
Conclusions
[0409] A novel class of PLGA/pSi microspheres was fabricated by an
S/O/W emulsion method by incorporating polymer science with
micro-lithography and electrochemical etching. This system provides
a number of unique advantages over pre-existing drug delivery
materials thanks to its ability to: 1) prevent the burst release of
proteins and prolong the delivery rate over a longer period of time
through the tuning of the PLGA coating; 2) counteract the
acidification of the environment by PLGA degradation byproducts via
buffering with degradation products of the pSi particles; 3)
preserve protein stability and half-life as the S/O/W method
prevents protein degradation during the fabrication process; 4)
control cellular internalization and protein accumulation by
increasing the particle diameter with PLGA coatings and controlling
biomolecular release based on PLGA properties, respectively.
PLGA/pSi microspheres cannot be internalized by cells due to their
size, which is particularly important for the delivery of growth
factors and proteins interacting with extracellular receptors. 5)
stimulate mineralization by promoting the deposition of calcium
phosphate ions on the particle surface. All together, these
findings demonstrate that the PLGA/pSi microspheres show superior
properties than traditional PLGA microspheres and represent a
promising alternative as drug delivery vehicles for tissue
engineering applications. Their use has been already successfully
tested in different orthopedic tissue engineering applications in
small and large animal models of bone fracture repair (manuscript
in preparation).
[0410] Experimental
[0411] pSi Particle Fabrication:
[0412] The pSi particles were fabricated as previously
described.[29] Briefly, an layer of silicon nitride (Si.sub.3N4)
(80 nm) was deposited by low pressure chemical vapor deposition on
a 4'' p-type Si wafer with resistivity <0.005. AZ5209
photoresist (AZ Electronic materials) was spun cast at 5000 R.P.M.
for 30 s on the substrate, followed by pre-exposure baking at
90.degree. C. in an oven for 10 min. A pattern consisting of dark
field circles (2 .mu.m) with pitch (2 .mu.m) was transferred on the
photoresist with a MA/MB6 mask aligner. The pattern was developed
for 20 s in MIF 726 developer, and then transferred into the
silicon nitride (Si.sub.3N4) layer and 300 nm into the silicon
substrate by two step Reactive Ion Etch (first step: Plasmatherm
790, 25 sccm CF.sub.4, 200 mTorr, 250 W RF, 2 min 20; second step:
Oxford Plasmalab 80, 20 sccm SF.sub.6, 100 mTorr, 200 W RF, 4 min).
The photoresist was removed from the substrate by an 8 min piranha
clean (H.sub.2O.sub.2:H.sub.2SO.sub.4 1:2 v/v). The porous
particles were formed by anodic etch in Hydrofluoric acid (HF):
ethanol (1:3 v/v) applying a current (0.3 A) for 60 s followed by
3.8 A for 6 s in a custom Teflon etching cell. The Si.sub.3N.sub.4
layer was removed by soaking in HF for 30 min, the substrate was
dried and the particles were released in isopropanol (IPA) (Acros)
by sonication.
[0413] Z2 Analysis and Surface Modification of pSi:
[0414] For oxidation, the dried pSi particles were resuspended in a
piranha solution and heated to 110-120.degree. C. for 2 h. The
suspension was washed with DI water until the pH was approximately
5.5-6.0. Oxidized pSi particles were suspended in ISOTON.RTM. II
Diluent, and counted by a Multisizer 4 Coulter.RTM. Particle
Counter (Beckman Coulter) with an aperture (20 .mu.m). PSi
particles were surface modified with APTES (Sigma Aldrich) as
reported previously. [16] 1.times.10.sup.8 oxidized particles were
suspended in of Millipore water (20 .mu.l). A solution was prepared
of 2.0% APTES and 3.0% Millipore water in IPA. This solution (980
.mu.l) was added to the particles and mixed well. This vial was
placed to a 35.degree. C. thermomixer set to mix at 1300 rpm for 2
h. After modification, the particles were washed with anhydrous IPA
5 times and moved to a vacuum oven for annealing at 60.degree. C.
overnight.
[0415] Loading of FITC-BSA into APTES Modified pSi Particles:
[0416] FITC-BSA (Sigma Aldrich) solution (10 mg/ml) was prepared by
dissolving FITC-BSA powder in distilled water. 4.times.10.sup.8
APTES modified particles were immersed into of FITC-BSA solution
(200 al) in an eppendorf tube. The suspension was incubated on a
thermal mixer at 37.degree. C. under agitation for 30 min to allow
the adsorption of the protein into the pores of pSi particles. The
particles were separated by centrifugation and washed with PBS to
remove the FITC-BSA physically absorbed on the surface. The
FITC-BSA loaded particles were then lyophilized overnight. The
amount of protein absorbed was measured by the difference between
the protein concentrations of the stock solution and of the
supernatant using SpectraMax M2 spectrophotometer (Molecular
Devices).
[0417] Preparation of PLGA Particles and PLGA Coated pSi
Particles:
[0418] pSi particles coated with PLGA were prepared by a modified
S/O/W emulsion method [47] as shown in FIG. 44. Briefly, PLGA
(50:50) (Sigma Chemicals Co. St. Louis, Mo.) was dissolved in
dicholoromethane (DCM) (Sigma Aldrich) to form 6%, 10%, and 20% w/v
PLGA/DCM solution respectively. 8.times.10.sup.7 FITC-BSA loaded
particles were suspended in these solutions (1 ml, 6%, 10%, and
20%) respectively by sonicating the mixture. The organic phase
containing the pSi was mixed with of Poly (vinyl alcohol) (PVA)
(Fisher Scientific) (3 ml, 2.5% w/v) by vortex mixing and
sonication. The mixture was gradually dropped into water (50 ml)
containing PVA (0.5% w/v). The resulting suspension was stirred
with a magnetic stir bar for 2 h and the DCM was rapidly eliminated
by evaporation. The PLGA/pSi microspheres were washed with
distilled water. Finally, the product was lyophilized and stored at
4.degree. C. PLGA particles were prepared in the similar method as
PLGA/pSi microsphere fabrication, except that BSA solution instead
of BSA loaded pSi particles was mixed with PLGA/DCM.
[0419] Characterization of PLGA/pSi Microspheres:
[0420] The morphology of the microspheres was characterized by
optical microscope (Nikon Eclipse TS 100), fluorescent microscope
(Nikon Eclipse TE 2000-E), confocal laser microscope (Leica MD
6000), and scanning electron microscope (SEM) (FEI Quanta 400 ESEM
FEG). The samples were analyzed by confocal laser microscope at 488
nm to identify the FITC-BSA loaded pSi. The microspheres were also
examined by SEM under a voltage of 3 KV. The samples were sputtered
with gold (20 nm) by a Plasma Sciences CrC-150 Sputtering System
(Torr International, Inc) before SEM analysis.
[0421] Sorting Procedure:
[0422] Several centrifugation steps, optimizing time and rotation
rate of each step, were performed to separate the PLGA/pSi
microspheres from the empty PLGA microspheres. Separation was
carried out by three centrifugation steps of 10 min each at 500,
1200 and 4500 rpm respectively with the Allegra X-22 Centrifuge
(Beckman Coulter Inc.). pSi particles conjugated with DyLight 549
NHS-Ester (Thermo Scientific) coated with PLGA were analyzed by
FACS (Becton Dickinson, FACSCalibur) before and after sorting
procedure to asses sorting efficiency.
[0423] Evaluation of FITC-BSA In Vitro Release:
[0424] 2.times.10.sup.7 FITC-BSA loaded PLGA/pSi microspheres were
dispersed in PBS (1 ml) at 37.degree. C. At predetermined time
intervals, the suspension was centrifuged (4500 rpm; 5 min), and
the supernatant (1 ml) was collected, and replaced with fresh PBS
(1 ml). The amount of BSA released was determined by analysis of
the collected supernatant using a spectrophotometer at 493/518 nm.
The suspension was also analyzed by FACS and the samples were
prepared by mixing NaCl solution (150 .mu.l) with suspension (5
.mu.l) removed from in vitro release samples.
[0425] Degradation Studies:
[0426] The in vitro degradation of the PLGA/pSi microspheres was
investigated by monitoring the surface morphology of the
microspheres and the pH of the degradation media. The pH level was
monitored using a pH meter (Denver Instrument UB-10), and the
surface morphology of the microspheres was examined by SEM.
[0427] BSA Stability Studies:
[0428] SDS-PAGE gel electrophoresis was performed to determine the
hydrolysis of BSA during the FITC-BSA release from PLGA/pSi. Color
Silver Staining Kit was used to stain the gel, Mark 12 (Invitrogen)
was used as standards. Supernatant (100 .mu.l) released from
PLGA/pSi microspheres (6%, 10%, 20%) collected on day 7 and day 14
was filtered by Amicon Ultra-0.5 ml centrifugal filter (Millipore
Ultracel-3 Membrane, 3 kDa) before SDS-PAGE.
[0429] In Vitro Mineralization Studies:
[0430] The osteogenic media were prepared by base media
(.alpha.-MEM media) (Invitrogen) containing Fetal bovine serum
(20%, FBS) (Invitrogen) supplemented with L-glutamine (1%), sodium
pyruvate (1%, Invitrogen), penicillin/streptomycin (1%,
Invitrogen), and osteogenic supplement. PLGA/PSi microspheres were
immersed in osteogenic growth medium. After 3, 8 and 21 day
incubation, the specimens were washed carefully with DI water, and
dried under vacuum overnight before characterization. PLGA
microspheres were used as control. The samples were analyzed by SEM
coupled with energy dispersive x-ray (EDX) for mineralization
studies.
[0431] In Vitro Internalization Studies:
[0432] 6,500 BMSCs were seeded into a 4 chamber tissue culture
treated glass slides. When the cells were 30% confluent, PLGA/pSi
microspheres containing 65,000 pSi particles were added into each
chamber. 65,000 pSi particles were used as control. After 0 h, 24
h, 48 h, and 120 h incubation, cells were washed with PBS and fixed
with 4% paraformaldehyde (PFA) for 10 min at room temperature. PFA
was removed and washed twice with PBS. Cells were permeabilized
with 0.1% Triton X for 10 min, and then blocked with BSA (1%) in
PBS for 30 min at room temperature. Triton X was removed, and cells
were incubated with Alexa Fluor 555 conjugated phalloidin in BSA
(1%) in PBS for 30 min. Cells were washed and incubated with DRAQ5
for 1 h. DRAQ5 was removed and prolong gold was added on the slides
to mount the sample.
[0433] In Vitro Cellular Uptake of FITC-BSA:
[0434] 40,000 HUVEC were seeded and cultured on a glass coverslip
in a 12 well plate with 500 million PLGA/pSi microspheres loaded
with FITC-BSA in a transwell on top of the cells. The media were
changed every 3 days. Cellular uptake of FITC-BSA released from the
PLGA/pSi microspheres was observed by confocal microscopy staining
cells with fluorescent phalloidin (actin filaments) and DRAQ5
(nuclei) after fixation (10% formaldehyde).
[0435] Confocal Microscopy Analysis:
[0436] Detection of the FITC-BSA loaded pSi particles was based on
autofluorescence using 488 excitation laser and the cells were
analyzed by using 561 and 632 excitation laser for pholloidin and
DRAQ5 respectively. Images were acquired using a Leica MD 6000
upright confocal microscope equipped with a 63.times. oil immersion
objective.
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[0484] Without further elaboration, it is believed that one skilled
in the art can, using the description herein, utilize the present
invention to its fullest extent. The embodiments described herein
are to be construed as illustrative and not as constraining the
remainder of the disclosure in any way whatsoever. While the
preferred embodiments have been shown and described, many
variations and modifications thereof can be made by one skilled in
the art without departing from the spirit and teachings of the
invention. Accordingly, the scope of protection is not limited by
the description set out above, but is only limited by the claims,
including all equivalents of the subject matter of the claims. The
disclosures of all patents, patent applications and publications
cited herein are hereby incorporated herein by reference, to the
extent that they provide procedural or other details consistent
with and supplementary to those set forth herein.
* * * * *