Polymeric Delivery Vehicles for Bone Growth Factors - American

Chapter 13

Polymeric Delivery Vehicles for Bone Growth Factors Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 11, 2018 at 20:37:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Lichun Lu, Susan J. Peter, Georgios N . Stamatas, and Antonios G. Mikos 1

Departments of Bioengineering and Chemical Engineering, Rice University, 6100 Main Street, Houston, TX 77005-1892

Recombinant human transforming growth factor-β1 ( T G F - β 1 ) was incorporated into biodegradable microparticles o f blends o f p o l y ( D L -lactic-co-glyeolic acid) (PLGA) and poly(ethylene g l y c o l ) ( P E G ) at 6 ng per m g microparticle. Fluorescein isothiocynate-labelled bovine serum albumin ( F I T C - B S A ) was co-encapsulated as a porogen. The effects o f PEG content (0, 1, or 5 wt%) and buffer p H (3, 5, or 7.4) on T G F - β 1 release kinetics and PLGA degradation were determined in vitro for up to 28 days. T G F - β 1 was released i n a multi-phasic fashion including an initial burst effect. Increasing the PEG content resulted in decreased cumulative mass o f released proteins. Aggregation o f FITC-BSA occurred at acidic buffer p H , w h i c h led to decreased protein release rates. PLGA degradation was also enhanced at 5% P E G , w h i c h was significantly accelerated at acidic p H conditions. Co-encapsulation o f T G F - β 1 w i t h F I T C -Dextran reduced the initial burst effect as compared to FITC-BSA. The T G F - β 1 released from PLGA/PEG rnicroparticles enhanced the proliferation and osteoblastic differentiation of marrow stromal cells cultured on poly(propylene fumarate) ( P P F ) substrates. T h e cells showed significantly increased total cell number, alkaline phosphatase ( A L P a s e ) activity, and osteocalcin production after 21 days, as compared to cells cultured under control conditions without TGF-β1. These results suggest that PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-β1, w h i c h may find applications in modulating cellular response during bone healing at a skeletal defect site.

M a n y afflictions require controlled delivery of therapeutic molecules for effective treatment.

Transforming growth factor-βΐ ( T G F - β Ι ) has been studied as a potential

Corresponding author.

124

© 2000 A m e r i c a n C h e m i c a l Society

Park and Mrsny; Controlled Drug Delivery ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

125 14

induction factor for bone tissue e n g i n e e r i n g . T G F - β Ι is a multifunctional protein that regulates many aspects o f cellular activity i n c l u d i n g cell proliferation, differentiation, and extracellular matrix metabolism, in a time- and concentrationdependent fashion . It plays a significant role in regulating bone formation at a fracture c a l l u s , and has been shown to increase osteoblast proliferation and differentiation during fracture healing in a rat femur m o d e l . Furthermore, T G F - β Ι was used to enhance healing in vivo in a canine total j o i n t replacement m o d e l . C o n t r o l l e d release o f T G F - β Ι to a bone defect may therefore be beneficial for the induction o f a bone regeneration cascade. 4

5

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Microparticles made o f biodegradable polymers have been w i d e l y utilized as vehicles for drug delivery. They can be implanted at an afflicted site during surgery or injected as a suspension to a w o u n d area. Alternatively, microparticles can be impregnated into polymer scaffolds and then transplanted . A m o n g different polymers, p o l y ( D L - l a c t i c - c o - g l y c o i i c acid) ( P L G A ) copolymers have been extensively studied as microparticle carriers for many bioactive molecules " . P L G A copolymers are biocompatible, biodegradable, and approved by the F D A for certain human clinical uses . Release rates o f bioactive molecules can be modulated by altering the molecular weight o f P L G A as w e l l as the ratio o f lactic to g l y c o l i c acid in the c o p o l y m e r . T h e combination o f poly(ethylene g l y c o l ) ( P E G ) and P L G A as a blend to form microparticle carriers allows further attenuation o f the release profile o f the loaded compound . In addition, the release o f compounds is also affected by coencapsulation o f other molecules in the microparticle formulations . Furthermore, the protein release profdes are also dependent o n environmental conditions such as the acidity o f the release m e d i u m . F o r example, the inflammatory response f o l l o w i n g implantation o f devices or the release o f acidic P L G A degradation products at late stages o f microparticle degradation often results in decrease in local p H . This decrease in p H can affect the structure, solubility, diffusivity, and activity o f the loaded compound. A n o v e l injectable, in situ polymerizable, biodegradable orthopaedic material based on poly(propylene fumarate) ( P P F ) for filling skeletal defects has recently been developed in our laboratory . P P F , combined w i t h a v i n y l monomer (/V-vinyl pyrrol id inone) and an initiator (benzoyl peroxide), forms an injectable paste that can be p o l y m e r i z e d in situ, f i l l i n g skeletal defects o f any shape or s i z e . A d d i t i o n a l l y , incorporation o f s o l i d phase components i n c l u d i n g β-tricalcium phosphate ( β - T C P ) and sodium chloride can result in a porous composite material possessing mechanical properties sufficient for the replacement o f human trabecular b o n e . Furthermore, marrow stromal cells were shown to attach, proliferate, and express differentiated 20 osteoblastic function when cultured on Ρ Ρ Ρ / β - T C P substrates in vitro . It is an objective o f our laboratory to develop an injectable formulation that may provide a vehicle for delivery o f microparticle carriers o f growth factors to the defect site and induce bone regeneration. 8

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1 0 , 1 6

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The present study addresses the effects o f P E G content and buffer p H on

the

release kinetics o f T G F - β Ι from biodegradable P L G A / P E G blend microparticles and the degradation o f P L G A ,

as w e l l as the effects o f T G F - β Ι

released from these

microparticles on the proliferation and differentiation o f primary rat marrow stromal cells seeded on P P F substrates.


126 Controlled Release of Transforming Growth Factor-βΐ from Biodegradable Polymer Microparticles Biodegradable Polymer Microparticles Microparticles o f blends o f poly(DL-lactie-co-glyeolic acid) ( P L G A ; M e d i s o r b ® , A l k e r m e s , C i n c i n n a t i , O H ) w i t h a 50:50 lactic to g l y c o l i c acid copolymer ratio and poly(ethylene g l y c o l ) ( P E G ; A l d r i c h , M i l w a u k e e , W I ) were fabricated using a doubleemulsion-solvent-extraction technique ((water-in-oil)-in-water) as previously described . P L G A had a weight average molecular weight ( M w ) o f 46,700 and a polydispersity index (PI) o f 1.73, as determined by gel permeation chromatography (GPC) . P E G w i t h a M w o f 10,700 was incorporated at 0, 1, and 5 w t % in the microparticles. Recombinant human transforming growth factor-βΐ ( T G F - β Ι ; R & D Systems, M i n n e a p o l i s , M N ) w i t h a molecular weight o f 25,000 was loaded at 6 ng per m g microparticle. Fluorescein isothiocynate-labeiled bovine serum a l b u m i n ( F I T C - B S A ; S i g m a , St. L o u i s , M O ) w i t h a molecular weight o f 68,000 was coencapsulated as a porogen at 4 μ g per m g microparticle. T h e average diameter o f the microparticles containing 0, 1, or 5 w t % initial P E G , reported as mean ± standard deviation ( S D ) based on normal distribution, was 20.0 ± 11.9, 18.8 ± 9.9, and 23.3 ± 13.7 μηι, respectively. The entrapment efficiency o f the proteins i n the microparticles was determined by n o r m a l i z i n g the amount actually entrapped to the starting amount, using an established solvent extraction technique . T h e entrapment yields o f T G F - β Ι were 83.4 (± 13.1), 84.6 (± 16.4), and 54.2 (± 12.1) % (n = 4) for P E G contents o f 0, 1, and 5 w t % , respectively. Single factor analysis o f variance ( A N O V A ) was used to assess statistical significance o f results. S c h e f f é ' s test showed the entrapment efficiency at 5% P E G was significantly lower (p < 0.05), w h i c h may be due to the leaching o f compounds w i t h soluble P E G during microparticle fabrication. 14,21

1

Scanning electron microscopy ( S E M ) revealed that the fabricated P L G A / P E G microparticles were spherical in shape, w i t h smooth, non-porous surfaces ( F i g . la). In addition, fluorescence images indicated fairly uniform distribution o f F I T C - B S A (and probably T G F - β Ι ) throughout the microparticles ( F i g . l b ) .

TGF-βΙ Release Kinetics The release kinetics o f T G F - β Ι from P L G A / P E G microparticles were studied under six experimental conditions. Microparticles w i t h varied initial P E G contents (0, 1, and 5 w t % ) were placed in p H 7.4 phosphate buffered saline ( P B S ) for up to 28 days. A d d i t i o n a l microparticles containing 5% P E G were incubated i n buffers o f p H 3, 5, or 7.4. A t the end o f various time points, the amounts o f T G F - β Ι in the releasing media were analyzed by enzyme-linked immunosorbent assay ( E L I S A ) . B o t h microparticle P E G content ( F i g . 2a) and m e d i u m acidity ( F i g . 2b) were found to affect the release of T G F - β Ι from the microparticles. After 2 days in P B S , 2.9 ± 0.2, 2,7 ± 0.3, and 1.9 ± 0.3 n g o f the c o m p o u n d per m g microparticle were released for initial P E G content o f 0, 1, and 5 wt%, respectively. W h i l e in buffers of


127

Figure /, Fabricated PLGA/PEG blend microparticles with 5 wt% PEG: (a) scanning electron micrograph and (b)fluorescencemicrograph. Scale bar is 20 pm for (b).


128

Figure 2. Cumulative release kinetics of TGF-βΙ (co-encapsulated with FITC-BSA) from PLGA/PEG microparticles for different (a) PEG content and (b) buffer pH. Error bars represent means ± SD for η = 4.


129 p H 3, 5, and 7.4, 0.6 ± 0.1, 0.9 ± 0.4, and 1.9 ± 0.2 ng o f T G F - β Ι per m g of microparticle were released from microparticles containing 5% P E G , respectively.

At

p H 7.4 (either P B S or p H buffer), the initial burst was followed by a linear phase of s l o w release rate reaching a plateau.

In acidic p H , however, the initial burst was

f o l l o w e d by a fast linear release phase (days 1 -8) and then by a slower one (days 9-28). T h e cumulative mass o f released T G F - β Ι was increased at lower P E G content or higher buffer p H . After 28 days, 3.4 ± 0.2 and 2.2 ± 0.3 ng o f loaded T G F - β Ι were released from microparticles with 0 and 5% P E G in p H 7.4 P B S , and 2.0 ± 0.2 and 1.3 ± 0.4 ng released for 5% P E G in p H 7.4 and 3 buffers, respectively.

In vitro degradation of microparticles T h e degradation o f P L G A / P E G microparticles (loaded w i t h T G F - β Ι and F I T C B S A ) were studied under the same conditions as in the protein release experiments. T h e M w o f P L G A , evaluated by G P C , decreased continuously throughout the time course for microparticles with varied P E G content placed in p H 7.4 P B S ( F i g . 3a). B y day 28, 35.6 ( ± 0.5), 34.4 ( ± 1.3), and 29.5 ( ± 1.5) % o f the day 0 M w remained for microparticles w i t h 0, 1, and 5 w t % P E G , respectively. The half-life o f P L G A , calculated by fitting the data for M w to an exponential function o f t i m e , was 15.9 ± 1.2 days for microparticles w i t h 5% P E G , significantly lower (p < 0.05) than that for 0 and 1% P E G (20.3 ± 0.9 and 18.9 ± 0.5 days, respectively). Degradation profiles o f P L G A microparticles w i t h 5% initial P E G content in various p H buffers are shown in F i g . 3b. B y day 28, the remaining M w o f P L G A placed in p H 3, 5, and 7.4 buffers was 3.1 (± 0.3), 14.0 (± 0.8), and 25.6 ( ± 4.6) % of the day 0 value, respectively. The corresponding half-lives o f P L G A were 5.5 ± 0 . 1 , 10.9 ± 0.4, and 14.8 ± 0.4 days, significantly dependent on the environmental p H (p < 0.01). Fluorescence images showed that the distribution o f F I T C - B S A was m a i n l y at the peripheral o f the microparticles after 14 days o f degradation in p H 7.4 buffers ( F i g . 4a). M o s t o f the protein was released by day 28 ( F i g . 4b). However, under acidic conditions such as p H 3, F I T C - B S A had limited solubility. T h i s led to aggregation o f F I T C - B S A in the polymer matrix, as confirmed by enhanced fluorescence after 14 days ( F i g . 4c). The aggregated F I T C - B S A formed a paste-like structure after complete P L G A degradation at 28 days ( F i g . 4d). The decreased release o f both T G F - β Ι and F I T C - B S A at lower p H is believed to result from this aggregation of insoluble compounds. 14

Effects of T G F - β Ι Released from Biodegradable P o l y m e r M i c r o p a r t i c l e s on M a r r o w S t r o m a l Osteoblast C u l t u r e

Biodegradable Polymer Substrates Poly(propylene fumarate) ( P P F ) was synthesized from fumaryl chloride and propylene g l y c o l in the presence o f potassium carbonate, a proton scavenger. The


130

50000-,

Time (days) Figure 3. Decrease of weight average molecular weight (Mw) of PLGA in PLGA/PEG microparticles for different (a) PEG content and (b) buffer pH. Error bars represent means ± SD for η = 4.


131

Figure 4. Fluorescence micrographs showing the distribution of FITC-BSA within PLGA/PEG microparticles with 5 wt% PEG after (a,c) 14 and (b,d) 28 days of in vitro degradation in (a,b) pH 7.4, and (c,d) pH 3 buffers. Scale bar is 20 pm.


132 resulted P P F (1 g) was then m i x e d w i t h i V - v i n y l pyrrolidinone (0.33 m L ) and b e n z o y l peroxide (0.005 g) in a c y l i n d r i c a l m o l d (1.8 c m diameter). C u r e d cylinders were sectioned into 1.5-mm thick disks. These discs, after leaching out unreacted material, d r y i n g , sterilization by ethylene oxide, and prewetting w i t h sterile P B S , were used as substrates for cell culture.

TGF-βΙ Release Kinetics T G F - β Ι was incorporated into P L G A / P E G blend microparticles w i t h 5 w t % P E G at 6 n g per m g microparticle. F I T C - D e x t r a n was co-encapsulated as a porogen at 1 w t % .

The entrapment efficiency o f T G F - β Ι

was 40.3

(± 1.2) % (n =

corresponding to an actual loading o f 2.42 n g per m g microparticle.

3),

The release

kinetics showed that 17.9 ( ± 0.6) and 32.1 (± 2.5) % o f loaded T G F - β Ι was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks ( F i g . 5).

F I T C - D e x t r a n a l l o w e d modulation o f T G F - β Ι release profiles w i t h a smaller

burst effect as compared to F I T C - B S A described in the previous section.

After 24 h ,

5 4 % o f the total released T G F - β Ι was freed from the microparticles, i n contrast to a value o f 7 5 % w h e n co-encapsulated w i t h F I T C - B S A .

Marrow Stromal Cell Function in Response to Released TGF-βΙ The femurs and tibias o f 6-week-old male Sprague-Dawley rats were harvested as previously described to isolate marrow stromal c e l l s . After 10 days o f expansion in tissue culture flasks, the marrow-derived cells were seeded at 24,000 c e l l s / c m onto tissue culture polystyrene ( T C P S ) and P P F substrates placed at the bottom of transwell plates. C o m p l e t e media containing Dulbecco's modified eagle m e d i u m ( D M E M ) supplemented w i t h 10% fetal bovine serum ( F B S ) , 20 penicillin/streptomycin/neomycin (PSN), 20 μg/mL fungizone, 1x10" M dexamethasone, 10 m M N a β - g l y c e r o l phosphate, and 50 μ g / m L L-ascorbic acid were added to each w e l l . The cells were subsequently maintained in complete m e d i a for 24 h to induce the osteoblastic phenotype o f the marrow stromal cells \" Microparticles w i t h encapsulated T G F - β Ι and F I T C - D e x t r a n were suspended i n primary media ( D M E M supplemented w i t h 10% F B S , 20 μ g / m L P S N , and 20 μ g / m L fungizone) and added to the top portion o f transwell plates. C e l l s seeded on P P F substrates and treated w i t h blank microparticles w i t h neither T G F - β Ι nor F I T C - D e x t r a n , as w e l l as cells seeded o n P P F and T C P S and cultured in the absence o f any microparticles served as controls. The effects o f T G F - β Ι released from P L G A / P E G microparticles on marrow stromal cell proliferation and function were assessed during a 21-day period. 22

2

Cell Number C e l l proliferation was assessed by measuring the total cell numbers using the D N A assay ( F i g . 6). The c e l l numbers increased rapidly for a l l sample sets through day 7, indicating high proliferative activity o f the cells. T h i s was followed b y a plateau for the rest o f the time course w i t h little cell proliferation. The sample sets exposed to released T G F - β Ι had significantly higher cell counts after day 7. B y day 24


133

Figure 5. Cumulative release kinetics of TGF-βΙ (co-encapsulated with FITCDextran) from PLGA/PEG microparticles into ρ H 7.4 PBS. Error bars represent means ± SD for η = 3.


134

150-,

m TCPS PPF PPF+Blank

• •

ο ο Ο

PPF+TGF-βΙ 100

s

Ζ

50

υ

Time(days) Figure 6. Total number of marrow stromal cells cultured on PPF substrates in the presence of TGF-βΙ-loaded PLGA/PEG microparticles during 21 days of in vitro culture. PPF substrates with blank microparticles, PPF, and TCPS served as controls. Error bars represent means ± SD for η = 3. Star indicates values significantly higher than all the control groups.


135 2 1 , the total number o f cells cultured on P P F in the presence o f T G F - β Ι had reached 138,700 ± 3,300 c e l l s / c m , w h i l e those for control sample sets T C P S , P P F , and blank microparticles were 123,700 ± 2,300, 114,300 ± 3,900 and 119,600 ± 6,100 c e l l s / c m , respectively. 2

2

Alkaline Phosphatase Activity C e l l function was monitored by determining the expression o f two markers of osteoblastic phenotype: alkaline phosphatase ( A L P a s e ) activity and osteocalcin p r o d u c t i o n . A t day 7, all sample sets had a moderate level o f A L P a s e activity, w i t h no difference in activity between sample sets ( F i g . 7). However, at both day 14 and 2 1 , the A L P a s e activity was significantly higher (p < 0.05) for the cells maintained in the presence o f T G F - β Ι than a l l control conditions. The A L P a s e activity for this sample set at day 21 was 22.8 (± 1.5) χ ΙΟ" μ η ι ο le/m in/cell, significantly higher than the next highest value o f 16.3 (± 2.9) χ ΙΟ" p m o l e / m i n / c e l l for the cells cultured on P P F w i t h blank microparticles. 24

7

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Osteocalcin Production T h e effect o f T G F - β Ι on osteocalcin production o f marrow stromal cells was s i m i l a r to that observed for A L P a s e activity. The cells exposed to T G F - β Ι had a significantly higher (p < 0.05) level of osteocalcin released into the m e d i u m than those maintained i n the absence o f T G F - β Ι ( F i g . 8), reaching 15.9 ( ± 1.5) χ 10"

6

ng/cell at this time point.

6

The control cultures had values o f 13.0 (± 1.0) χ 10" 6

ng/cell for T C P S substrates, 12.5 (± 1.6) χ 10" ng/cell for P P F substrates, and 12.2 6

(± 1.2) χ 10" ng/cell for the P P F cultures exposed to blank microparticles.

Conclusions T G F - β Ι was encapsulated along with F I T C - B S A into P L G A / P E G blend microparticles and released i n a controlled fashion in vitro for up to 28 days. Increasing the initial P E G content resulted in lower entrapment efficiency o f T G F - β Ι and decreased cumulative mass o f released compound. Protein release was decreased under acidic p H due to aggregation o f F I T C - B S A . The degradation o f P L G A was increased at higher P E G content, and significantly accelerated at lower buffer p H . C o encapsulation with F I T C - D e x t r a n resulted in reduced initial burst effect o f released T G F - β Ι . The T G F - β Ι released from the microparticles enhanced the proliferation and osteoblastic differentiation o f marrow stromal cells cultured on P P F substrates, as indicated by the increased total cell number, A L P a s e activity, and osteocalcin production over a period o f 21 days.

Acknowledgments T h i s w o r k was completed through funding provided by the N a t i o n a l Institutes of Health ( R 0 1 - A R 4 4 3 8 1 ) . S.J. Peter acknowledges financial support by the National Institutes o f Health B i o t e c h n o l o g y T r a i n i n g Grant 5 T 3 2 G M 0 8 3 6 2 .


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Time (days) Figure 7. Alkaline phosphatase (ALPase) activity of marrow stromal cells cultured on PPF substrates in the presence of TGF-fil-loaded PLGA/PEG microparticles during 21 days of in vitro culture. PPF substrates with blank microparticles, PPF, and TCPS served as controls. Error bars represent means ± SD for η - 3. Star indicates values significantly higher than all the control groups.


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14

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Time (days) Figure 8, Osteocalcin production of marrow stromal cells cultured on PPF substrates in the presence of TGF-fil-loaded PLGA/PEG microparticles daring 21 days of in vitro culture. PPF substrates with blank microparticles, PPF, and TCPS served as controls. Error bars represent means ± SD for η = 3. Star indicates values significantly higher than all the control groups.


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