as) unitedstates a2) patentapplicationpublication 10) pub ... · m pog(n=4) —_——__— less.17...
TRANSCRIPT
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US 20140141047A1
as) United States
a2) Patent Application Publication 10) Pub. No.: US 2014/0141047 Al
Murphyet al. (43) Pub. Date: May22, 2014
(54) SELECTION OF PLATELET RICH PLASMA Publication Classification
COMPONENTSVIA MINERAL BINDING(51) Int. Cl.
(71) Applicant: WISCONSIN ALUMNI RESEARCH AGIK 47/48 (2006.01)
FOUNDATION,Madison, WI (US) (52) U.S. CL.
CPC viececceccseescssenseenecnees AGIK 47/48992 (2013.01)
(72) Inventors: William L. Murphy, Waunakee, WI USPC viccccccscesseseessssesstssssstssnesteanees 424/400; 514/1.1(US); Ray Vanderby, Madison, WI
(US); Geoffrey Baer, Verona, WI (US); (57) ABSTRACTBen K. Graf, Madison, WI (US); Jae . . oo,Sung Lee, Madison, WI (US); Connie Mineral coated devices and methods for delivering an autolo-
Chamberlain, Madison, WI (US) gousbiological molecule using the mineral coated devices are
disclosed. The mineral coated devices allow for the isolation(73) Assignee: WISCONSIN ALUMNI RESEARCH and delivery of a biological molecule obtained from the same
FOUNDATION,Madison, WI (US) subject to avoid the safety concerns of current biologicaltherapies obtained by recombinant methods and purification
(21) Appl. No.: 13/680,251 from animal sources. Methods for selectively isolating and
(22) Filed: Nov. 19, 2012
eluting a biological molecule using the mineral coated
devices are also disclosed.
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Patent Application Publication May 22,2014 Sheet 1 of 20 US 2014/0141047 Al
FIG. 1A
ANAKSN S N\A SS
x ~~ .
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US 2014/0141047 AlMay 22, 2014 Sheet 2 of 20Patent Application Publication
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Patent Application Publication May 22,2014 Sheet 3 of 20 US 2014/0141047 Al
FIG. 3A
2 8000 -.
Off Curve O <5 min 1 30 min
7000 7 7 60 min 11 120 min6000 F
5000 F 1
WS4000
3000
2000
1000 P
ROTEINCONCENTRATION(ng/
0 ! Z
1 0DILUTION FACTOR OF PRP
0 <5 min 4 30 min
1000 N 60 min O 120 min
> 800} Tc 7 T~ = IT
5 600 Uf; SY
5E 400+LL
SOo 200°Oo
0 J
PRP DILUTION FACTOR:10
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Patent Application Publication May 22,2014 Sheet 4 of 20 US 2014/0141047 Al
FIG. 4
= 120- 41 30 min9S
2 100 IN 60 min
i 1 120 min-—
80-
:= 60+OS 40+ -Oo> po rT _a 20 RS
° 0 v | ot [ere
a. 1 10 100
DILUTION FACTOR
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FIG.
5
PLATELETRICHPLASMA
(1HOUR)
1011
«12
001M
PO4(
n=4)
.17M
PO4(
n=4)
.25M
PO4(
n=4)
AL
LpH=7.4
MINERALCOATED
PO4ELUTION:
WASH
|-05MPO4(n=4)
15,60,90
mineS
—=
.11MPO4(n=4)
——»BCAASSAYTO
MEASUREPROTEIN
MEAURETOTAL
THATDETACHED
PROTEIN
WITH
PO4
CONCENTRATION
Patent Application Publication May 22, 2014 Sheet 5 of 20 US 2014/0141047 Al
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Patent Application Publication May 22,2014 Sheet 6 of 20 US 2014/0141047 Al
FIG. 6
307A 015 min : b,c
E257 T |> b,c 1 I
LL LLz 20 | b
E ist :9Qa
Ee 10°FZz
O
mT ;| | | | |
0.001 0.05 0.11 0.17 0.25
p<.0001 PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 7 of 20 US 2014/0141047 Al
FIG. 7
40
35
30
25
20
15
10
060 min
ao
HHS
b C
i
HHA
I
oT
Is
+H
T
How
PERCENTPROTEINELUTED
0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 8 of 20 US 2014/0141047 Al
FIG. 8
aA 357. Cc
- Lm ool | b,c
25 b 1
= Tt IE 20+ |
a 15+
Z 10+
2 5st 2Lu Lo
oO 0 l l J
0.001 0.05 0.11 0.17 0.25
p<.0001 PHOSPHATE BUFFER MOLARITY (M)
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FIG.
9
HCI/NaOH
DISS
OLUT
IONOF
H20(n
=4)
PO4EL
UTIO
N:MI
NERA
L.001
MPO
4(n=
4)WASH
|
-05M
PO4(
n=4)
<5,
30,90
min
HCl/
NaOH
—~
4.11MPOg(n=4)
—_——__—
LESS
.17M
PO4(
n=4)
MEAS
UREPR
OTEI
NME
ASUR
EPROTEIN
.29M
PO,
(n=4)
THATDETACHED
BOUNDTOMINERAL
MINERAL
4WITHPO4
ALLpH=7.4
\/
MINERALCOATED
AGILENTASSAYTO
MEASURETOTAL
PROTEIN
CONCENTRATION
PLATELETRICHPLASMA
(1HOUR)
2.03
X10°PLATELETS/{u
lWit
iz
Patent Application Publication May 22, 2014 Sheet 9 of 20 US 2014/0141047 Al
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Patent Application Publication May 22,2014 Sheet 10 of 20 US 2014/0141047 Al
3—~
2 14-S 12+ | O<5 min
F 40+ |
= |E 8+fa2 | T |S 4b L l
= 2+5 0 l yy l l l | l
o H20 0.001 0.05 01 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
=2 2.9
O<5 minSa2 15} ] ]
g |O i1F |
8 [ | !> 057
to5 0 ! ! ! ! |
ae H20 0.001 0.05 0Q.11 O17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 11 of 20 US 2014/0141047 Al
FIG. 11A
= 30 -= 030 min=> 25 |
: |2O-
©E ist |O tZ 10} + =O
zraa H20 0.001 0.05 O11 40.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
a G) . 11B
250 -
1130 min200 +
150 -
100 + —++— +4
50+
0 l \ oo l l l
H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
PROTEINCONCENTRATION(ng/
scl)
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Patent Application Publication May 22,2014 Sheet 12 of 20 US 2014/0141047 Al
FIG. 12A
1190 min
15-
10 +
7 L T .
0 | | | | | a |
H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
PROTEINCO
NCEN
TRAT
ION(ng/zl)
FIG. 12B
e N9
oO |
100 1190 min
80
i i60 | t
40 +
so |—_E]l. oo, |
H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
©
PROTEINCONCENTRATION(ng/jl)
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FIG.
13
MINERALCOATED
PRP+H20
(CX;
n=4)
PRP+
.01MPO
4(n=
4)PRP+
.05MPO
4(n=
4)PR
P+
.11MPO
4(n=
4)
UNBOUNDPR
OTEI
NSBOUNDPROTEINS
15,30
,90mi
n(-———HCI/NaOH
SS0
1i—-
MEASUREPROTEIN
MEASUREPROTEIN
PRP+
.17MPO4(n=4)
PRP+
.25MPOg(n=4)
THATDI
DN'T
BIND
BOUNDTOMINERAL
WITHPO4
ALLpH=7.4
\/
AGILENTPROTEIN
ASSAY
Patent Application Publication May 22, 2014 Sheet 13 of 20 US 2014/0141047 Al
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Patent Application Publication May 22,2014 Sheet 14 of 20 US 2014/0141047 Al
FIG. 14
310,2 9fF 115 minZ 8Fr |
et | |fe SF || | ih
BST LJO 4bS 3b i lz= 2hEe ifo 0 | | | | | |
oa H20 0.001 0.05 O41 O17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 15 of 20 US 2014/0141047 Al
FIG. 15
2—o)
& 12,z 030 min
S 10} ISs 8 IE otZ 6-4
Q | T= 4+ TL T IOo T tO 2- T
2 tL
td 0 | | | | | J
O H20 0.001 £0.05 0.11 0.17 0.25
Qa PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication
FIG. 16
PROTEINCONCENTRATION(ng/
sel) = AN
— NO
_ Oo
kL
D@®
+
May 22, 2014 Sheet 16 of 20
b—_+—
US 2014/0141047 Al
L190 min
HH
H20 0.001 0.05 0.11 0.17
PHOSPHATE BUFFER MOLARITY (M)
0.25
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US 2014/0141047 Al2014 Sheet 17 of 20May22,Patent Application Publication
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Patent Application Publication
FIG. 18A
BSACONCENTRATION(
ng/zz1)
9080 +70605040302010
May 22, 2014 Sheet 18 of 20 US 2014/0141047 Al
oO <5 min
HH
FIG. 18B
BSACONCENTRATION(
ng/zu!)
H20 0.001 0.05 0.41 O47 0.25
PHOSPHATE BUFFER MOLARITY (M)
/—}—4
Tr
L
oO<5min
/—}—
ih, H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 19 of 20 US 2014/0141047 Al
FIG. 19A
60 -
50+ O©15min
40+ L
30+
20 +
10 +
HH
HH
+—|
BSACONCENTRATION(
ng/;
z!)
H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
FIG. 19B
—_—
© oO l
1015 min80 -
olwo |!
H+4
BSACONCENTRATION(
ng/;z)
© l l l | |_|H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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Patent Application Publication May 22,2014 Sheet 20 of 20 US 2014/0141047 Al
FIG. 19C
BSACONCENTRATION(
ng/;
z1) 90 -
80 L <@coo BSA unbound
70 b <=BSAbound to mineral
H20 0.001 0.05 0.11 0.17 0.25
PHOSPHATE BUFFER MOLARITY (M)
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US 2014/0141047 Al
SELECTION OF PLATELET RICH PLASMACOMPONENTSVIA MINERAL BINDING
STATEMENT OF GOVERNMENT SUPPORT
[0001] This invention was made with governmentsupportunder AR059916 and HLO093282 awarded by the National
Institutes of Health. The governmenthascertain rights in theinvention.
BACKGROUNDOF THE DISCLOSURE
[0002] The present disclosure relates generally to medical
devices. More particularly, the present disclosure relates tomedical devices having a mineral coated substrate and an
autologous biological molecule. The present disclosure fur-ther relates to methods for selectively isolating a biological
molecule from a bodily fiuid using the coated devices and
methods for selectively eluting a biological molecule fromthe coated devices.
[0003] The delivery of biological molecules (“biologics”)such as growth factors to promote musculoskeletal healing
has become a popular approach in industry, with the market
for orthopedic growth factors, for example, nearly quadru-pling between 2003 and 2008. Onedelivery strategy involves
embedding biologics within collagen sponges for insertioninto a tissue defect. This strategy has been usedclinically for
delivery of the biologic BMP2 andis under development fordelivery of other emerging biologics, such as BMP 12.
[0004] Despite the clinical success of biologics, there are
significant limitations related to their delivery. For example,the materials that serve as carriers for biologics, such as
collagen sponges, are often inappropriate for orthopedicapplications because they do not seamlessly incorporate into
standard clinical procedures, and thus, require adoption and
training ofthe medicalpractitioner.In addition, biologic mol-ecules may quickly diffuse away from carrier materials and
mayrapidly degrade in vivo (e.g., t;,. of BMP2~minutes),whichresults in limited bioavailability and a need to deliver
large doses of the biological molecules. These large dosesmay be costly and maypresenta significant safety concern in
the clinical orthopedics community,as they have led to edema
and ectopic bone formation in multiple recent clinical studies.Thus, there are significant challenges associated with devel-
oping biologics for clinical applications.
[0005] Bloodis a biological therapy that may be used forwhole blood transfusions or making medications. Medica-
tions produced from specific portions ofthe whole blood maybe, for example, plasma,platelets, red blood cells, and white
blood cells.
[0006] Allogeneic (or homologous) blood transfusion usesbloodthat is collected from a blood donorandis used for the
transfusion of another subject. A specific blood type must bematched for safe transfusion. Another common blood dona-
tion practice is for a subject to have blood withdrawn inanticipation of needing blood (..e., self-donation). If, for
example, a subject is planning to undergo surgery where a
blood transfusion may be necessary, the subject may haveblood withdrawn andstored prior to the procedure. Autolo-
gous donation may eliminate reactions due to donor-recipientincompatibility and may preclude exposure to transfusion
transmitted infection.
[0007] Use of platelet rich plasma therapy is an emergingbiologic treatment that may influence the healing of tissues.
Platelet rich plasma (“PRP”) is blood plasma that has been
May22, 2014
enriched with platelets. Platelet enrichmentinvolvesthe col-lection ofwhole bloodthat is anticoagulated before undergo-
ing centrifugation to separate PRP from platelet-poor plasmaand red bloodcells. In humans, the baseline platelet concen-
tration of whole blood is about 160-370 k/ul. PRP containsabout 4-10x concentration ofbaseline platelet concentration.
The healing influence of PRP maybeattributed to the supra-
physiological concentrations of growth factors that arereleased by activated platelets. Many of the growth factors
contained in PRP have been identified as possible biologicsand studies are under wayto isolate and develop these growth
factors for use as biologics.[0008] Recent studies indicate that PRP may accelerate
healing, especially in tissues having a poor blood supply. For
example, PRP administration improved filling and biome-chanical testing of partial anterior cruciate ligamenttears.
PRP administration has also been shown to increase failureforce for Achilles tendon injury and stimulate the develop-
mentofnew bone and tendon in infraspinatus model. Clinical
use of PRP has produced promising, but inconsistent resultsdue to the broad variability in the production of PRP by
various concentrating equipment and techniques, as well asindividualvariability in the platelet concentration of plasma.
[0009] The preparation and use of biologics, especiallythose such as blood and blood-derived products, may involve
a cumbersomeregulatory path. Many growth factors used as
biologics are prepared using recombinant protein expressionmethods, which must undergo regulatory approval. For
example, approval of biologics by the U.S. Food and DrugAdministration and the European Medicines Agency involves
showing the safety, purity, potency andefficacy ofa biologic.Blood used for transfusions also undergoesa significant bat-
tery of tests to avoid the transmission of blood-borne patho-
gens.[0010] Accordingly, there exists a need to develop materi-
als and methods for isolating biological molecules such asblood and blood components for therapeutic applications.
SUMMARY OF THE DISCLOSURE
[0011] The present disclosure relates generally to materialsand methods for isolating and delivering biological mol-
ecules from bodily fluids. Moreparticularly, the present dis-closure relates to coated devices that can deliver biological
molecules isolated from bodily fluids such as, for example,
PRP andPRP components. The present disclosure also relatesto methods for isolating and eluting biological molecules
from bodily fluids. The coated devices and methods may beused in the operating room, for example, prior to or during a
surgical procedure, to isolate biological molecules from abodily fluid from a patient.
[0012] Thecoated devices and methodsoffer the possibility
of selecting specific biological molecules from bodily fluidssuch as, for example, blood and blood-derived solutions that
maybe obtainedintraoperatively. Moreover, because the bio-logical molecules may be autologous biological molecules,
the dosing and regulatory issues facing current biological
molecules may be mitigated.
[0013] In one aspect,the present disclosure is directed to a
coated device for delivering an autologous biological mol-ecule having a mineral coating on a substrate and an autolo-
gous biological molecule.
[0014] In another aspect, the present disclosure is directedto a method for selectively isolating a biological molecule
from a bodily fluid. The method includes preparing a coated
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US 2014/0141047 Al
device having a mineral coating on a substrate. The coateddevice is then incubated with a bodily fluid comprising a
biological molecule, wherein the bodily fluid further com-prises an ionic buffer.
[0015] In another aspect, the present disclosure is directed
to amethodfor selectively eluting a biological molecule froma coated device. The method includes preparing a coated
device having a mineral coating ona substrate; incubating thecoated device with a bodily fluid; and eluting the biological
molecule from the coated device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The disclosure will be better understood, and fea-
tures, aspects and advantagesother than those set forth above
will become apparent when consideration is given to thefollowing detailed description thereof. Such detailed descrip-
tion makes reference to the following drawings, wherein:
[0017] FIG. 1A is a scanning electron micrograph of a
mineralized poly lactide glycolide (PLG)film as discussed inExample 1.
[0018] FIG. 1B is a scanning electron micrograph of a
mineralized PLG film at a higher magnification as discussedin Example 1.
[0019] FIG. 2 is a schematic depicting binding ofplateletrich plasma (PRP) components to mineralized PLG films as
analyzed in Example 1.
[0020] FIG. 3A is a graphillustrating the total protein con-centration of diluted PRP incubated with the mineralized
PLGfilm as analyzed in Example 1.
[0021] FIG.3Bisascaled up version ofthe graph showninFIG.3A illustrating the total protein concentration of the 10
dilution incubated with the mineralized PLGfilm as analyzedin Example 1.
[0022] FIG. 4 is a graph illustrating the time- and dose-dependent changes in PRPprotein bindingto the mineralized
PLGfilms as analyzed in Example 1.
[0023] FIG. 5is a schematic depicting the selective elutionof PRP as analyzed in Example2.
[0024] FIG. 6 isa graphillustrating the amountofprotein
eluted from mineral coated wells after exposure to varyingPO, concentrations for 15 minutes as analyzed in Example2.
[0025] FIG. 7 isa graphillustrating the amountofproteineluted from mineral coated wells after exposure to varying
PO, concentrations for 60 minutes as analyzed in Example2.
[0026] FIG. 8 is a graphillustrating the amountofproteineluted from mineral coated wells after exposure to varying
PO, concentrations for 90 minutes as analyzed in Example 2.
[0027] FIG. 9 is a schematic depicting the selective elution
of PRP as analyzed in Example3.
[0028] FIG. 10A is a graphillustrating the amountofpro-tein eluted from mineral coated wellsafter a time point ofless
than 5 minutes using varying PO, concentrations as analyzed
in Example 3.
[0029] FIG. 10B is a graph illustrating the amountofpro-
tein bound to mineral coated wells after exposure to varyingPO, concentrations as analyzed in Example 3.
[0030] FIG. 11Ais a graphillustrating the amountofpro-
tein eluted from mineral coated wells after a time point of 30minutes using varying PO, concentrations as analyzed in
Example 3.
[0031] FIG. 11B is a graph illustrating the amountofpro-tein bound to mineral coated wells after exposure to varying
PO, concentrations as analyzed in Example 3.
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[0032] FIG. 12A is a graphillustrating the amountofpro-tein eluted from mineral coated wells after a time point of 90
minutes using varying PO, concentrations as analyzed inExample 3.
[0033] FIG. 12B is a graph illustrating the amountofpro-tein bound to mineral coated wells after exposure to varying
PO, concentrations as analyzed in Example 3.
[0034] FIG. 13 is a schematic depicting the selective bind-ing of PRP as analyzed in Example 4.
[0035] FIG. 14 is a graphillustrating the amountofproteinbound to a mineralized PLG film coating after a 15 minute
concomitant exposure to PRP and PO, buffer as analyzed inExample 4.
[0036] FIG. 15 is a graphillustrating the amountofprotein
boundto a mineralized PLG film after a 30 minute concomi-tant exposure to PRP and PO,buffer as analyzed in Example
4.[0037] FIG. 16 is a graphillustrating the amountofprotein
boundto a mineralized PLG film after a 90 minute concomi-
tant exposure to PRP and PO,buffer as analyzed in Example4.[0038] FIG. 17 is a schematic depicting the selective elu-tion ofbovine serum albumin (BSA)as analyzed in Example
5.[0039] FIG. 18A is a graphillustrating the amount ofBSA
eluted from a mineralized PLGfilm after exposure to varying
PO, concentrations for less than 5 minutes as analyzed inExample 5.
[0040] FIG. 18B is a graphillustrating the amount ofBSAboundto a mineralized PLG film after exposure to varying
PO, concentrations for less than 5 minutes as analyzed inExample 5.
[0041] FIG. 19A is a graphillustrating the amount ofBSA
eluted from a mineralized PLGfilm after exposure to varyingPO, concentrations for 15 minutes as analyzed in Example5.
[0042] FIG. 19B is a graphillustrating the amount ofBSAboundto a mineralized PLG film after exposure to varying
PO, concentrations for 15 minutes as analyzed in Example5.[0043] FIG. 19C is a graph illustrating the relationship
between bound and unbound BSAafter a 15 minute exposure
to varying PO, concentrations as analyzed in Example 5.
[0044] While the disclosure is susceptible to various modi-
fications and alternative forms, specific embodiments thereofhave been shown by wayofexample in the drawings and are
herein described below in detail. It should be understood,
however, that the description of specific embodiments is notintended to limit the disclosure to cover all modifications,
equivalents and alternatives falling within the spirit and scopeof the disclosure as defined by the appendedclaims.
DETAILED DESCRIPTION
[0045] Unless defined otherwise, all technical and scien-tific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which thedisclosure belongs. Although any methods and materials
similar to or equivalent to those described herein may be used
in the practice or testing of the present disclosure, the pre-ferred materials and methods are described below.
[0046] In accordance with the present disclosure, coateddevices and methodsfor selectively binding and eluting bio-
logical molecules from bodily fluids using the coated devices
have been discovered. Methods using coated devices as dis-closed herein provide a high throughputplatform for selec-
tively binding a desired biological molecule as well as a high
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US 2014/0141047 Al
throughput platform for determining a desired release profileof a biological molecule from the coated device. Signifi-
cantly, the coated device having an autologous biologicalmolecule of the present disclosure permits the delivery of a
biological molecule obtained from the same subject. Thisfeature avoids significant safety concerns with biological
molecules prepared using traditional methods such as, for
example, recombinant methods and isolation methods fromanimal sources. The methods further allow for the selective
isolation and selective elution of specific biological mol-ecules in a bodily fluid such that specific biological molecules
may be delivered to a subject from the coated devices of thepresent disclosure.
Coated Devices with a Mineralized Substrate and an Autolo-
gous Biological Molecule
[0047] In one aspect,the present disclosureis directed to acoated device for delivering an autologous biological mol-
ecule. The coated device has a mineral coating on a substrateand an autologous biological molecule attachedthereto. Suit-
able coated devices may be, for example, an orthopedicdevice, a particle, a film, a dish, a plate, and a suture. Particu-
larly suitable orthopedic devices may be, for example, an
arrow,a barb, a tack, an anchor, a nail, a pin, a screw, a staple,a plate, and combinations thereof. Particularly suitable par-
ticles may be, for example, agarose beads, latex beads, mag-netic beads, and combinations thereof. Particularly suitable
plates may be, for example, microtiter plates having, forexample, 6, 14, 96, or more sample wells.
[0048] The coated device includes a mineral coating on the
surface of a substrate. Suitable mineral coatings are madefrom mineral forming materials such as, for example, cal-
cium, phosphate, carbonate, and combinations thereof. Moreparticularly, as described morefully herein, the mineral coat-
ings may be formed on the substrate by surface hydrolyzing
the substrate underalkaline conditions. After surface hydro-lyzing, the substrate is incubated in a modified simulated
body fluid (mSBF) containing a suitable mineral-formingmaterial. Suitable substrates for use with the coatings may be,
for example, a poly(a-hydroxyester). Particularly suitablepoly(a-hydroxyesters) may be, for example, poly(L-lactide),
poly(lactide-co-glycolide), poly(e-caprolactone), and combi-
nations thereof.
[0049] Attachedto the coating, is an autologousbiological
molecule. The autologous biological molecule is obtained
from an autologousbodily fluid. The term “autologous bodilyfluid” is used hereinto refer to a bodily fluid that is obtained
from a subject and used as the source of the autologousbiological molecule, which is attached to the coated device
that is returned to the same subject. Thus, the subject is boththe donor and recipient of the autologous biological mol-
ecule. For example, if the autologous bodily fluid is platelet
rich plasma (PRP), the PRPis obtained from a subject and isthen incubated with the coated device according to the
method described herein to bind an autologous biologicalmolecule contained within the subject’s own PRP. Suitable
autologous bodily fluids may be, for example, whole blood,serum, plasma, platelet rich plasma, bone marrow, cere-
brospinal fluid, urine, synovial fluid, and combinations
thereof.
[0050] Suitable autologous biological molecules obtained
from the autologous bodily fluids may be proteins, for
example. Particularly suitable proteins may be, for example,basic proteins. The term “basic protein” is used herein
according to its ordinary meaning as understood by those
May22, 2014
skilled in the art to refer to the category of proteins that have
ahigh isoelectric point(pI offrom about 7.1 to about 14), and
therefore, tend to be positively charged at physiological pH(~7.4). By contrast, the term “acidic protein” is used herein
according to its ordinary meaning as understood by those
skilled in the art to refer to the category of proteins that havea low isoelectric point (pI of from about 0 to about 6.9), and
therefore tend to be negatively charged at physiological pH(~7.4). Particularly suitable basic proteins may be, for
example, growth factors.
[0051] Suitable growth factors may be, for example, bone
morphogenic proteins (BMPs), connective tissue growth fac-tors, epidermal growth factors, fibroblast growth factors
(FGFs), insulin-like growth factors, interleukin, keratinocyte
growth factors, platelet-derived growth factor (PDGFs),transforming growth factors (TGFs), vascular endothelial
growth factors (VEGF), nerve growth factor (NGF), hepato-cyte growth factor (HGF), tumor necrosis factors (TNF),
interferons (IFN), and combinationsthereof. Specific suitablegrowth factors may be, for example, BMP1, BMP2, BMP3,
BMP4, BMP5, BMP6, BMP7, BMP8a, EGF, PDGFA,PDGFB, PDGFC, PDGFD, PDGFAB, VEGF-A, placentagrowth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, TGF-61, TGF-B2, TGF-63, AMH, ARTN, GDF1, GDF2, GDF3,GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10,GDF11, GDF15, GDFN, INHA, INHBA, INHBB, INHBC,INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRIN,PSPN, IL-1, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, IL-21,IL-23, IL-17, IFN-a, IFN-y, TNF-a, TNF-6, FGF1, FGF2,FGF3, and FGF4.
[0052] The resulting coated device having an autologousbiological molecule bound thereto may then be administered,
for example, back into the same subject that served as thedonorofthe autologous bodily fluid. For example, the coated
device may be implanted in a subject to deliver the autologousbiological molecule to the subject.
[0053] Because the coated device of the present disclosurehas an autologous biological molecule, the coated device
allows for the delivery of the autologousbiological molecule
without the concerns associated with using biological mol-ecules obtained by traditional methods such as, for example,
recombinant methods and from animal sources. Also,because the coated devices have a mineral coating that is
degradable, delivery of the autologous biological moleculesmaybe controlled such as through controlled elution of the
biological molecule from the mineralized coating and/or con-
trolled degradation of the mineral coating. As the mineralcoating degrades, the attached autologous biological mol-
ecule is released from the coated device. Moreover, theautologous biological molecule attached to the coated device
may stimulate repair and/or growth by stimulating cells sur-roundingorrecruited to the area containing the coated device.
Additionally, therapeutically effective amounts ofthe autolo-
gousbiological molecule may be administered as the concen-tration of the autologous biological molecules on the coated
device may be controlled.
Methodsfor Selectively Isolating a Biological Molecule from
a Bodily Fluid
[0054] In one aspect,the present disclosure is directed to a
methodforselectively isolating a biological molecule from abodily fluid. The method includes preparing a coated device
comprising a mineral coating on a substrate and incubating
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US 2014/0141047 Al
the coated device with a bodily fluid comprising a biologicalmolecule, wherein the bodily fluid further comprises an ionic
buffer.
[0055] To prepare the coated device, a mineral coating may
first be formed on the substrate using methods described in
US. Patent Application Pub. No. 20080095817, U.S. Pat. No.8,075,562, and U.S. Patent Application Pub. No.
20110305760, which are hereby incorporated by reference tothe extent they are consistent herewith. For example, the
mineral coating may be formed by surface hydrolyzing asubstrate under alkaline conditions such as, for example,
NaOH,followed by incubation in a modified simulated body
fluid (mSBF) at a physiologic temperature and pH 6.8 formineral nucleation and growth. The mSBFsolution contains
mineral-forming ions, including calcium, phosphate, carbon-ate, and combinationsthereof. The resulting coating may be
any suitable coating material containing calcium, phosphate
and carbonate, such as, for example, hydroxyapatite (HAP),a-tricalcitum phosphate (a-TCP), 63-tricalcium phosphate
(B3-TCP), amorphous calcium phosphate, dicalcium phos-phate, octacalctum phosphate, and calcium carbonate. Fur-
ther, the coating formed on the substrate develops as a porousmineral coating (see FIGS. 1A and 1B). Although porous
mineral coatings are particularly suitable, the mineral coat-
ings may also be nonporous.
[0056] As described above, suitable substrates may be, for
example, a poly(a-hydroxyester). Particularly suitable poly(a-hydroxy esters) may be, for example, poly(L-lactide),
poly(lactide-co-glycolide), poly(e-caprolactone), and combi-
nations thereof.
[0057] The coated device having the mineral coated sub-
strate is then incubated with a bodily fluid including one ormore desired biological molecules. In one embodiment, the
bodily fluid may include autologousbodily fluid as described
above.
[0058] Alternatively, the bodily fluid is a heterologous
bodily fluid. As used herein, the term “heterologous bodilyfluid” (1.e., non-autologous solution) refers to a bodily fluid
that is obtained from one subject and used in the method to
prepare coated device having a biological molecule attachedthereto. The resulting coated device is then used for treating a
different subject(i.e., not the subject that donated the bodilyfluid). Thus, a subject who is the donor of the heterologous
bodily fluid used in the methodis different from a subject who
is the recipient of the coated device. The term “heterologousbodily fluid”alsorefers to a bodily fluid that is obtained from
a different species ofanimal, which is then used in the methodfor isolating a biological molecule of the present disclosure.
For example, a bodily fluid such as PRP from a sheep may beused to isolate a biological molecule from the sheep PRP,
whichis then used for a non-sheep animal as the recipient.
[0059] Suitable autologous and heterologous bodily fluidsmay be, for example, whole blood, serum, plasma, platelet
rich plasma, bone marrow, cerebrospinal fluid, urine, syn-ovial fluid, and combinationsthereof.
[0060] The bodily fluid further includes an ionic buffer.
Advantageously, including an ionic buffer in the bodily fluidallowsfor the selective binding ofbiological moleculesto the
coated device. The ionic buffer may be added to the bodilyfluid while the coated device is incubated. Without being
boundby theory, adding an ionic buffer to the bodily fluid
during incubation with the coated device may interfere withthe formation of electrostatic interactions between the min-
eral coating and the biological molecule contained in the
May22, 2014
bodily fluid. For example, if the ionic buffer added to thebodily fluid is high enough to prevent the formation of elec-
trostatic interactions between a biological molecule and themineral coating, the biological molecule may notbindto the
mineral coating. Thus, the ionic strength of the buffer that isadded to the bodily fluid influences binding of a biological
molecule to the mineral coating. For example, the ionic buffer
may influence binding of a biological molecule such that thebiological molecule does not interact at all to the coated
device, weakly interacts with the coated device, and/orstrongly interacts with the coated device.
[0061] Suitable ionic buffers that may be used in the
methodforselectively isolating a biological molecule from abodily fluid may be any ionic buffer that disrupts, interferes
with, and/or competes with the electrostatic interaction
between the biological molecule and the mineral of the min-eral coating on the coated device. Particularly suitable ionic
buffers may be, for example, phosphate buffers, sodium chlo-ride buffers, magnesium chloride buffers, calctum chloride
buffers, sodium fluoride buffers, and combinations thereof.Particularly suitable phosphate buffers may have a phosphate
concentration up to, and including, 0.5 M phosphate, and
including from about 0.001 M to 0.5 M phosphate. Particu-larly suitable sodium chloride buffers may have a sodium
chloride concentration up to, and including, 0.2 M sodiumchloride. Particularly suitable magnesium chloride buffers
may have a magnesium chloride concentration up to, and
including, 5.0 M magnesium chloride. Particularly suitablecalcium chloride buffers may have a calcium chloride con-
centration up to, and including, 5.0 M calcium chloride. Par-ticularly, suitable sodium fluoride buffers may have a sodium
fluoride concentration up to, and including, 0.4 M sodiumfluoride.
[0062] Suitable ionic buffers may be, for example, buffers
having varying pH ranges. Suitable pH ionic buffers mayhave a pH range of from about 6.4 to about 7.8.
[0063] The coated device having the mineral coated sub-
strate is incubated with the bodily fluid fora sufficient period
of time to allow attachment of a biological molecule to themineral coating of the substrate. Suitable time to allow the
biological molecule to attach to the mineral coating of thesubstrate may be, for example, from less than a minute to
about 120 minutes. The biological molecule attaches to themineral coating by electrostatic interactions.
[0064] The method mayfurther include washing the coated
device to remove components contained within the bodily
fluid that do not bind to the mineral coating. Washing mayremoveserum albumin, for example.
[0065] Thecoated device may be washed using anysuitable
washing solution. Suitable washing solutions may be, forexample, HEPES (4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid), saline, 0.001 M phosphate buffer, double dis-
tilled water, and combinationsthereof.
[0066] Attachmentof a biological molecule may be moni-tored using methods knownto those skilled in the art. For
example, the total protein concentration of the bodily fluidbefore and after incubation with the coated device may be
monitored using a BCA (bicinchoninic acid) assay. Othersuitable assays that may be used to monitor attachment and/or
elution maybe, for example, ELISA, Western blot, 1D and2D
SDS-PAGE, non-equilibrium pH gel electrophoresis(NEPHGB), AGILENT™protein analysis, and combinations
thereof.
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US 2014/0141047 Al
[0067] The coated devices resulting from the methods may
be coated devices having a mineral coating on a substrate and
a heterologous or autologous biological molecule attached
thereto. If the resultant coated device is one having a heter-
ologous biological molecule, the coated device is used for a
subject that is different from the subject that donated the
bodily fluid (the heterologous bodily fluid) used in the
methodto selectively isolate the biological molecule. Alter-
natively, ifthe resultant coated device is one having an autolo-
gous biological molecule, the coated device is used for a
subject that also was the subject that donated the bodily fluid
(the autologous bodily fluid) used in the method to selectively
isolate the biological molecule.
[0068] The resultant coated device may be administered toa subject. The resultant coated device may be administered,
for example, as an implant. For example, the resultant coated
device may be implantedin a subjectto deliver the biologicalmolecule to a subject.
[0069] The resultant coated device having an autologous
biological molecule allows forthe delivery ofthe autologous
biological molecule without the concerns associated withusing biological molecules obtained by traditional methods
such as, for example, recombinant methods and isolationmethods from animal sources. Thus, the resultant coated
device having an autologous biological molecule can avoidregulatory hurdles and safety issues associated with biologi-
cal molecules obtained from sources other than from the
subject’s own bodily fluids. The resultant coated device hav-ing a heterologous biological molecule allowsfor the delivery
of the heterologous biological molecule under conditionswherethe recipient subject may not have a sufficient amount
ofa biological molecule such that the recipient can also be the
donorof the bodily fluid used in the method. Additionally, aresultant coated device having a heterologous biological mol-
ecule may be suitable for use in a veterinary setting, whereone donor subject may be used to provide the bodily fluid
used in the method for preparing multiple coated devices orwhere having an autologous biological molecule is not
desired.
[0070] Because the coated devices have a mineral coating
that is degradable, delivery ofthe autologousbiological mol-ecule and the heterologous biological molecule may be con-
trolled such through controlled elution of the biological mol-
ecule from the mineral coating as described herein and/orcontrolled degradation ofthe mineral coating. As the mineral
coating degrades, the attached autologous biological mol-ecule and/or the heterologous biological molecule may be
released from the coated device. Additionally, or alterna-tively, as the ionic concentration ofthe environmentin which
the coated device is implanted changes, it may influence the
electrostatic interaction between the biological molecule andthe mineral coating such that the biological molecule
detaches from the mineral coating.
[0071] Moreover, the autologous biological molecules and
the heterologous biological molecules may stimulate repairand/or growth by stimulating cells surroundingorrecruited to
the area containing the coated device. Therapeutically effec-tive amounts ofthe autologousbiological molecule and/or the
heterologousbiological molecule may be administered as the
concentration ofthe autologous biological molecules and theheterologous biological molecule on the coated device may
be controlled.
May22, 2014
Methodsfor Selectively Eluting a Biological Molecule froma Coated Device
[0072] In another aspect, the present disclosure is directedtoa methodfor selectively eluting a biological molecule from
a coated device. The method includes preparing a coated
device having a mineral coating on substrate; incubating thecoated device with a bodily fluid having a biological mol-
ecule; and eluting the biological molecule from the coateddevice. Eluting may remove biological molecules that are
attached to the mineral coating on the substrate, and in someembodiments, allows for selectively removing a biological
molecule from the coated device.
[0073] In one aspect, eluting at least one biological mol-ecule from the coated device includes contacting the coated
device with an elution buffer. Suitable elution buffers may beany ion-containing buffer that disrupts the electrostatic inter-
action betweenthe biological molecule and the mineral ofthe
mineral coating on the coated device. Particularly suitableelution buffers that may be usedto elute at least one biological
molecule from the coated device may be, for example, phos-phate buffers (up to 0.5 M phosphate), sodium chloride buff-
ers (up to 0.2 M sodium chloride), magnesium chloride buff-ers (up to 5.0 M magnesium chloride), calcium chloride
buffers (up to 5.0 M calcium chloride), sodium fluoride buff-
ers (up to 0.4 M sodium fluoride), and combinationsthereof.
[0074] Selective elution of the biological molecule may be
performed as a “batch-type” elution in which the coateddevice is contacted with a particular ionic strength buffer such
that elution of the biological molecule occurs at once. Selec-
tive elution ofthe biological molecule mayalso be performedusing a concentration gradient in which the beginning elution
is performed using a low ionic strength buffer and continueswith increasing ionic strength buffer. The gradient may be
performedas a step-wise gradientor as a continuousgradient.
[0075] In another aspect, eluting at least one biologicalmolecule from the coated device includes contacting the
coated device with a mineral dissolution buffer. The mineraldissolution buffer causes the mineral ofthe mineral coating to
dissolve. As the mineral coating dissolves, a biological mol-ecule that is electrostatically attached to the mineral coating
losesits attachment and elutes from the coated device. Suit-
able mineral dissolution buffers may be any buffer that causesthe mineral coating to dissolve.
[0076] Particularly suitable mineral dissolution buffers
may include phosphoric acid, ethylenediaminetetraaceticacid (EDTA), 0.25 M hydrochloric acid (HCl), 0.25 M
sodium hydroxide (NaOH), for example. The mineral disso-lution buffer may include phosphoric acid up to, and includ-
ing, 0.5 M phosphoric acid. The mineral dissolution buffermay include EDTA upto, and including, 20% (w/v) EDTA.
The amount of HCl in the mineral dissolution buffer may be
up to, and including, 0.25 M HCl.
[0077] Elution of a biological molecule may be monitored
using methods knownto thoseskilled in the art. For example,the total protein concentration of the bodily fluid before and
after incubation with the coated device as well as after the
coated device is contacted with an ionic buffer or a mineraldissolution buffer may be monitored using a BCA assay.
Othersuitable assays that may be used to monitor elution maybe, for example, ELISA, Western blot, 1D and 2D SDS-
PAGE,non-equilibrium pH gel electrophoresis (NEPHGE),AGILENT™protein analysis, and combinations thereof.
[0078] The disclosure will be more fully understood upon
consideration of the following non-limiting Examples.
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EXAMPLES
Example 1
Mineralized Film and PRP
[0079] In this Example, the protein concentration of PRP
wasdetermined after incubation with a mineralized poly lac-
tide glycolide (PLG)film.
[0080] Specifically, poly lactide glycolide (85:15) wassol-
vent casted into a film. The film was mineralized for 10 daysusing mSBF to form a mineralized coating on the PLG film
(FIG. 1A and FIG. 1B). Blood wascollected from sheep and
centrifuged at 312xg and 1248xg to obtain PRP having aplatelet count of 886 k/ul. PRP was subjected to cycles of
freeze-thaw to lyse platelets. The resulting PRP was dilutedby factors of 1, 10, and 100 in 0.001 M phosphate (PO,)
buffer. The total protein concentration of each PRP dilutionwas determined by BCA assay.
[0081] As shown in FIG. 2, mineralized PLG films were
incubated with each PRP dilution at 0 minute, 30 minute, 60minute and 120 minute time points to allow proteinsto bind to
the mineralized PLG films. After incubation, the films weretransferred to a new plate and rinsed with 0.001 M PO,buffer
to remove unboundprotein. The PRP solution from which the
films were transferred was collected and used to measureunboundprotein. The bound proteins were then eluted from
the mineralized PLGfilms using 0.2 MNaOH andneutralizedusing 0.2 M HCl in 0.01 M HEPES.Protein concentration of
(1) PRP before addition to the mineralized PLG films, (2)
PRPafter incubation with the mineralized PLGfilms, and (3)0.2 M NaOHeluate were determined using a BCAassay.
[0082] As shown in FIG.3A and FIG.3B,thetotal proteinconcentration ofPRP decreased asthe dilution increased. As
shown in FIG. 4, incubation of PRP with mineralized PLG
films resulted in binding ofproteins to the mineralized PLGfilms. It was also possible to elute the protein that was bound
to the mineralized PLG film. Ofthe three dilutions, the con-dition thatresulted in the most total bound protein wasby the
mineralized PLG film incubated for 120 minutes with PRPthat was diluted by a factor of 10.
Example 2
Selective Elution of PRP Components
[0083] In this Example, elution of proteins from mineral-
ized PLG wells with varying phosphate molarities was deter-mined
[0084] Specifically, wells of a 96-well plate were coated
with a mineralized film made as described above. PRP wasobtained and subjected to cycles of freeze-thaw to lyse plate-
lets as described above. As shownin FIG.5, mineralized PLGwells were incubated for 1 hour to allow proteins to bind.
Unbound proteins were rinsed off using water. Bound pro-
teins were eluted from the mineralized PLG wells for 15minute, 60 minute, and 90 minute time points using 0.001 M,
0.05 M,0.11 M, 0.17 M, and 0.25 M PO,(1°Protein Elution)and measured by BCA assay. Proteins that remained bound
after the 1°’ Protein Elution were eluted from the mineralizedPLG wells by incubating the mineralized PLG wells using 0.2
M NaOH.The 0.2 M NaOHeluate wasneutralized using 0.2
M HClin 0.01 M HEPES (2”” Protein Elution). The proteinconcentration of the 2”“ Elution was measured using a BCA
assay.
May22, 2014
[0085] As shownin FIG.6 for the 15 minute timepoint, aconcentration of 0.001 M PO, eluted the least amount of
proteins, whereas 0.05 M, 0.11 M, 0.17 M, and 0.25 M PO,eluted more proteins.
[0086] As shown in FIG.7 for the 60 minute timepoint, a
concentration of 0.001 M PO, eluted the least amount ofproteins, whereas 0.05 M, 0.11 M, 0.17 M, and 0.25 M PO,
eluted more proteins.
[0087] As shownin FIG.8 for the 90 minute timepoint, aconcentration of 0.001 M PO, eluted the least amount of
proteins, whereas 0.05 M, 0.11 M, 0.17 M, and 0.25 M PO,eluted more proteins.
Example 3
Selective Elution of PRP Components
[0088] In this Example, elution of proteins from mineral-
ized PLG wells with varying phosphate molarities was deter-
mined
[0089] Specifically, PLG wells were mineralized as
described above. PRP wasobtained and subjectedto cycles of
freeze-thaw to lyse platelets as described above. As shown inFIG.9, mineralized PLG wells were incubated for 1 hour to
allow proteins to bind. Unbound proteins were rinsed offusing water. Boundproteins were eluted from the mineralized
PLG wells for less than 5 minutes (buffers were placed intothe wells and immediately collected), 30 minutes and 90
minutes using water, 0.001 M, 0.05 M, 0.11 M, 0.17 M, and
0.25 M PO, (1°Protein Elution; PO, eluted) and measuredusing an AGILENT™protein assay. Proteins that remained
boundafter the 1°’ Protein Elution (using phosphate buffer)were eluted a second time from the mineralized PLG wells by
incubating the mineralized PLG wells using 0.2 M HC1/0.01M HEPES.The 0.2 M HC1/0.01 M HEPESeluate was neu-
tralized using 0.2 MNaOH (2”Protein Elution; HCI eluted).
The protein concentration of the 2”“ Elution was measuredusing an AGILENT™assay.
[0090] As shown in FIGS. 10A and 10Bfor the <5 minute
time point, mostofthe protein elutedin the 1°’ Protein Elutionas comparedwith the percentprotein eluted in the 2”“ Elution.
Additionally, as shown in FIG. 10A, both water and 0.011 MPO,eluted the least amountofproteins, whereas 0.05 M,0.11
M,0.17 M,and 0.25 M PO,eluted moreproteins. As shown
in FIG. 10B, however, after mineral dissolution via HCl, bothwater and 0.001 M PO, bound the most proteins, whereas
0.05 M, 0.11 M, 0.17 M, and 0.25 M PO, boundlessproteins.
[0091] As shown in FIG. 11A for the 30 minute timepoint,both water and 0.011 M PO,eluted the least amountofpro-
teins, whereas 0.05 M, 0.11 M, 0.17 M, and 0.25 M PO,eluted more proteins. As shown in FIG. 11B, however, after
mineral dissolution via HCl, both water and 0.001 M PO,boundthe most proteins, whereas 0.05 M, 0.11 M, 0.17 M,
and 0.25 M PO,boundless proteins. This data also indicated
that proteins exposed to PO, for 30 minutes have sufficienttimeto fully elute from the mineral coating. As shown in FIG.
12A for the 90 minute time point, 0.011 M PO, eluted themost amount of proteins, whereas 0.05 M, 0.11 M, 0.17 M,
and 0.25 M PO,eluted less proteins. As shown in FIG. 12B,however, after mineral dissolution via HCI, both water, 0.001
M and 0.05 M PO, boundthe mostproteins, whereas 0.11 M,
0.17 M, and 0.25 M PO, boundless proteins. This data alsoindicated the ability of proteins to bind andrelease at lesser
concentrations from lower density mineral coated wells.
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Example 4
Selective Binding of Proteins from PO,-ModifiedPRP
[0092] In this Example, binding of proteins in PO,-modi-
fied PRP to mineralized PLP films was determined
[0093] Specifically, PLG films were mineralized as
described above. PRP wasobtained and subjectedto cycles of
freeze-thaw to lyse platelets as described above. Varying con-centrations of phosphate (0.001 M, 0.05 M,0.11 M, 0.17 M,
and 0.25 M PO,) were added to the PRP to form the PO,-modified PRP. No phosphate was added to control PRP
(“Cx”). As shown in FIG. 13, mineralized PLP films wereincubated with Cx and modified-PRP for 15 minute, 60
minute and 90 minute time points. After the 15 minute, 30
minute and 90 minute incubation periods, the buffer (1° Elu-tion) was collected and analyzed using anAGILENT™assay
to measure total unboundprotein. Proteins that bound to themineralized PLG films were eluted with 0.2 N HCl (2”4
Elution) to release boundproteins. The solution was neutral-ized with 0.2 N NaOHandproteinsthat selectively bound to
the mineralized PLG film were thereby measured using an
AGILENT™assay.
[0094] As shown in FIG. 14 for the 15 minute time point,
both water, 0.001 M and 0.05 M PO,boundthe mostproteins,
whereas 0.11 M, 0.17 M and 0.25 M PO, boundless proteins.
[0095] As shown in FIG. 15 for the 30 minute time point,
both water and 0.001 M PO, bound the most proteins,whereas 0.05 M, 0.11 M, 0.17 M and 0.25 M PO,boundless
proteins.
[0096] As shown in FIG. 16 for the 90 minute time point,both water and 0.001 M PO, bound the most proteins,
whereas 0.05 M, 0.11 M, 0.17 M and 0.25 M PO,boundless
proteins.
Example 5
[0097] In this Example, binding of bovine serum albumin
(BSA) to mineralized PLG films was determined
[0098] As shown in FIG. 17, BSA was added to mineralcoated films and allowed to bind for 1 hour. Unbound BSA
wasrinsed away. PO, buffer at one of the following concen-
trations, 0.001 M, 0.05 M, 0.11 M, 0.17 M, and 0.25 M, wasadded to the mineralized films and allowed to incubate for
less than 5 minutes (buffer was placed into wells and imme-diately collected) and at a 15 minute time point. The buffer
solution was then collected to measure proteins that elutedfrom the mineralized film. The mineralized film was then
dissolved using 0.2 N HCIto release the boundproteins. The
solution was neutralized with 0.2 N NaOHandproteins thatselectively bound to the mineralized film were thereby mea-
sured using an AGILENT™assay.
[0099] Asshown in FIG.18A,at the less than 5 minute timepoint, concentrations of 0.001 M and 0.05 M PO,eluted the
least amountofproteins whereas 0.11 M, 0.17 M, and 0.25 MPO, eluted more proteins. As shown in FIG. 18B, water and
concentrations of 0.001 M and 0.05 M PO, bound the most
amount ofBSA proteins and 0.11 M, 0.17 M, and 0.25 M PO,boundless proteins.
[0100] As shown in FIG. 19A,at the 15 minute time point,
concentrations of 0.001 M and 0.05 M PO, eluted the leastamountofproteins whereas 0.11 M, 0.17 M, and 0.25 M PO,
eluted more proteins. As shown in FIG. 19B, water and con-
May22, 2014
centrations of 0.001 M and 0.05 M PO, bound the mostamount ofBSAproteins and 0.11 M, 0.17 M, and 0.25 M PO,
boundless proteins.
[0101] FIG. 19C illustratesthe relationship ofbound versusunbound BSA after a 15 minute exposure to varying PO,
concentrations. BSA boundto the mineral coating in a PO,-dose dependent manner. Combining twodifferent assays, it
has been shownthat: 1) there is reduced binding to mineralcoating with increasing PO, molarity (solid line); and 2) there
is increased free protein with increasing PO,, molarity (dotted
line). Taken together, these results indicate that albumin bind-ing to mineral is controlled via PO, concentration.
[0102] The examples described above demonstrate that the
mineral coatings and methodsoffer the ability to select spe-cific biological molecules from bodily fluids that may be
obtained intraoperatively. This will allow for the isolation ofa specific biological molecule or set of biological molecules
from a subject, then delivery of the specific biological mol-ecule back into the same subject or into a different subject.
[0103] In view of the above,it will be seen that the several
advantagesofthe disclosure are achieved and other advanta-geousresults attained. As various changes could be made in
the above devices and methods without departing from the
scope ofthe disclosure,it is intended that all matter containedin the above description and shown in the accompanying
drawings shall be interpretedasillustrative and notin a lim-iting sense.
[0104] When introducing elements of the present disclo-
sure or the various versions, embodiment(s) or aspectsthereof, the articles “a”, “an”, “the” and “said”are intended to
mean that there are one or more of the elements. The terms
“comprising”, “including” and “having” are intended to beinclusive and mean that there may be additional elements
other than the listed elements.
Whatis claimedis:
1. A coated device for delivering an autologous biologicalmolecule comprising a mineral coating on a substrate and an
autologous biological molecule attached to the mineral coat-
ing.
2. The coated device of claim 1, wherein the mineral coat-
ing is selected from the group consisting of calcium, phos-
phate, carbonate, and combinationsthereof.
3. The coated device of claim 1, wherein the substrate
comprises a poly(a-hydroxyester) selected from the group
consisting of poly(L-lactide), poly(lactide-co-glycolide),poly(e-caprolactone), and combinationsthereof.
4. The coated device of claim 1, wherein the autologous
biological molecule is isolated from an autologous bodilyfluid.
5. The coated device of claim 1, wherein the autologous
biological molecule is a protein.
6. The coated device of claim 5, wherein the protein is abasic protein.
7. The coated device of claim 6, wherein the basic protein
is a growth factor selected from the group consisting ofa bonemorphogenic protein, a connective tissue growth factor, an
epidermal growth factor, a fibroblast growth factor, an insu-lin-like growth factor, interleukin, keratinocyte growthfactor,
aplatelet derived growth factor, a transforming growth factor,
vascular endothelial growth factor, nerve growth factor(NGF), hepatocyte growth factor (HGF), tumornecrosis fac-
tors (TNF), interferons (IFN), and combinationsthereof.
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US 2014/0141047 Al
8. The coated device of claim 1, wherein the device isselected from the group consisting of an orthopedic device, a
particle, a film, a dish, a plate, and a suture.9. A methodforselectively isolating a biological molecule
from a bodily fluid, the method comprising:
preparing a coated device comprising a mineral coating ona substrate;
incubating the coated device with a bodily fluid comprisinga biological molecule, wherein the bodily fluid further
comprises an ionic buffer.
10. The method of claim 9, wherein the bodily fluid isselected from the group consisting of whole blood, serum,
plasma, platelet rich plasma, bone marrow, cerebrospinalfluid, urine, synovial fluid, and combinationsthereof.
11. The method of claim 9, wherein the ionic buffer com-
prises at least one ofphosphate, sodium chloride, magnesiumchloride, and calcium chloride.
12. The method ofclaim 11, wherein the ionic buffer com-prises up to 0.5 M phosphate.
13. The method ofclaim 9, wherein the substrate comprises
apoly(a-hydroxyester) selected from the group consisting ofpoly(L-lactide), poly(lactide-co-glycolide), poly(e-caprolac-
tone), and combinations thereof.
14. The method of claim 9, wherein the mineral coating is
selected from the group consisting of calctum, phosphate,
carbonate, and combinationsthereof.
15. A methodforselectively eluting a biological molecule
from a coated device, the method comprising:
May22, 2014
preparing a coated device comprising a mineral coating ona substrate;
incubating the coated device with a bodily fluid comprisinga biological molecule; and
eluting the biological molecule from the coated device.16. The method ofclaim 15, wherein eluting the biological
molecule from the coated device comprises contacting the
coated device with an ionic buffer selected from the groupconsisting of a phosphate buffer, a sodium chloride buffer, a
magnesium chloride buffer, a calcium chloride buffer, asodium fluoride buffer, and combinations thereof.
17. The method ofclaim 15, wherein eluting the biologicalmolecule from the coated device comprises contacting the
coated device with a mineral dissolution buffer, wherein the
mineral dissolution buffer is selected from the group consist-ing of phosphoric acid, ethylenediaminetetraacetic acid,
hydrochloric acid, sodium hydroxide, and combinationsthereof.
18. The method of claim 17, wherein the mineral dissolu-
tion buffer comprises up to 0.5 M phosphoric acid.19. The method ofclaim 15, wherein the mineral coatingis
selected from the group consisting of calctum, phosphate,carbonate, and combinationsthereof.
20. The method of claim 15, wherein the substrate com-prises a poly(a-hydroxy ester) selected from the group con-
sisting ofpoly(L-lactide), poly(lactide-co-glycolide), poly(e-
caprolactone), and combinationsthereof.
* * * * *