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The Van Houten library has removed some of the personal information and all signatures from the approval page and biographical sketches of theses and dissertations in order to protect the identity of NJIT graduates and faculty. ABSTRACT
CHARACTERIZATION OF ELECTROSPUN DLPLGA NANOFIBERS FOR A DRUG DELIVERY SYSTEM FOR INTRACRANIAL TUMORS AND AVMs
by Kimberly A. Griswold
E ec os i i g ocesses a y e ec ic ie s o a o yme so u io i o e o o uce s a s o o yme i e a osca e a ge ese o yme i e s a e ma i u a e o
ei o osi y ig su ace a ea i e ess a u i o mi y I o e o c ea e e i ea
ug e i e y sys em o i ac a ia a e io e ous ma o ma io s (A Ms a umo s
o y-( ac ice- o y-g yco i e ( GA a o i e s we e e ec os u i a
e a y o u a ( so e ee i e e co ce a io a ios o GA 5/15
/ a 75/ 5 we e a a y e o o ai e ime ase o ug a e a io 1 3- is ( - c o oe y -1- i osou ea ( C U was e co isso e wi e GA a o i e s
o o m a omoge ous so u io e ec os u a a a y e o ug c a ac e i a io
e SEM a a ysis s owe a i e iame e is o a u c io o ug ese ce
e GA g a s s owe i e a ia io i mass oss i com a i g e e ec os u i e
o e ug com i e i e e SC a a ysis o i e a g a ue a ou -53°C o
o e aw o yme a e e ec os u i e is o i e oo a e e ec os i i g ocess oes o a ec e c emica a u e o e o yme e ese ce o C U o e o yme (c ose / -1 w was e e mi e y e c a ge i g o se e o e SC g a s om a a ue o -53°C o wo a ues o 1 9°C a
1 9 °C CHARACTERIZATION OF ELECTROSPUN DLPLGA NANOFIBERS FOR A DRUG DELIVERY SYSTEM FOR INTRACRANIAL TUMORS AND AVMs
by Kimberly A. Griswold
A Thesis Submitted to the Faculty of New Jersey Institute of Technology In Partial Fulfillment of the Requirements for the Degree of Master of Science in Biomedical Engineering
Department of Biomedical Engineering
January 2004
APPROVAL PAGE
CHARACTERIZATION OF ELECTROSPUN DLPLGA NANOFIBERS FOR A DRUG DELIVERY SYSTEM FOR INTRACRANIAL TUMORS AND AVMs
Kimberly A. Griswold
Mic ae a e esyA iso a e esea c o esso o ioma e ia s I
C a es es igiaeOmo esis Co-A iso a e Me ica oc o a Assis a o esso o eu o ogica Su ge y a a io ogy UM
Mic ae ua g Commi ee Mem e a e Associa e o esso o C emica E gi ee i g I
ee a A i e Commi ee Mem e a e Assis a o esso o iome ica E gi ee i g I BIOGRAPHICAL SKETCH
Author: Kim e y A G iswo
Degree: Mas e s o Scie ce
Date: a ua y
Undergraduate and Graduate Education:
• Mas e o Scie ce i iome ica E gi ee i g Co ce a io i o yme Scie ce a ioma e ia s ew e sey I s i u e o ec o ogy ewa k
• ac e o o Scie ce i iome ica E gi ee i g Co ce a io i O ics U i e si y o oc es e oc es e Y
Major: iome ica E gi ee i g Co ce a io i o yme Scie ce a ioma e ia s Dedicated to the memory of my grandfather, Augustus W. Griswold, co-founder of Dynak Incorporated, engineer, innovative thinker, and inventor. Also to my devout and patient family and friends who through enlightenment allowed me to be comfortable in my own skin. Finally, to my Korean family, all inclusive, known and unknown - for the gift of life, new and old, and for anonymity.
v ACKNOWLEDGMENT
I would first like to recognize the support and guidance of Dr. Michael Jaffe and Dr.
Charles J. Prestigiacomo in not only my academic endeavors, but my personal ones as well. Dr. Michael Jaffe served as my research advisor, but was very influential in the academic path chosen and the career choices I have made. Dr. Charles J.
Prestigiacomo served as my assistant research advisor and provided constant support, encouragement, and intellectual stimulation to keep me motivated throughout the project.
Special Thanks goes to Dr. Michael Huang for providing such an amazing academic experience and guiding me to a particular field of concentration for this thesis research. It is he to whom I owe all my newfound interest and my desire to obtain greater knowledge in polymer science.
Appreciation to my fellow graduate students in the Medical Device and
Concept Laboratory, particularly Shobana Shanmugasundaram for her assistance and knowledge in the electrospinning process, and fine-tuned SEM and computer technique. Finally, particular acknowledgment to Joseph Pickton for his judgment and knowledge of Polymer Science, his support and strong shoulder to lean on, and his keen wit for humor.
vi
A E O CO E S
Ch pt r
1 I O UC IO I
1 1 O ec i e I
I ackg ou I o ma io
I 1 I ac a ia ascu a o ma io s 3
I Cu e ea me Me o s 7
1 3 a maki e ics o C U 11
1 io eg a a e o yme s 13
1 5 Ki e ics o ug e ease I5
1 GA C a ac e is ics
E EC OS I I G EC IQUE 9
3 MA E IA S 33
ME O O OGY 37
1 E ec os i i g ocess 37
C a ac e i a io o E ec os u i e s 3
3 E ec os i i g a C a ac e i a io o ug a o yme Com e
5 ESU S I
ISCUSSIO 73
7 CO C USIO 7
A E I A C A AC E IS ICS O ES EC I E A IOS O A A GA 7
A E I IOCOM A I I I Y O AC I E/G YCO I E CO- O YME S 79
vii TABLE OF CONTENTS (Continued)
Chapter Page
APPENDIX C MECHANICAL PROPERTIES OF MEDISORB® POLYMERS... 81
APPENDIX D SOLUBILITY CHART OF MEDISORB® POLYMERS 82
APPENDIX E THERMAL ANALYSIS DATA OF MEDISORB® POLYMERS 83
APPENDIX F CHEMICAL PROPERTIES OF BCNU 84
APPENDIX G GMP ANALYSIS OF 75/25 DLPLGA POLYMER 87
APPENDIX H GMP ANALYSIS OF 85/15 DLPLGA POLYMER 88
APPENDIX I NON-GMP ANALYSIS OF 80/20 DLPLGA POLYMER 89
APPENDIX J POLYMER CHARACTERISTICS OF MEDISORB® POLYMERS 90
APPENDIX K ENVIRONMENTALLY SENSITIVE DRUG RELEASE SYSTEMS 91
REFERENCES 92
iii LIST OF TABLES
Table Page
3 1 o uc I o ma io 33
5 1 i e O se a io s Usi g OM 3
5 a o i e iame e
5 3 OM i e O se a io s I
5 5/15-15w eco e iame e
5 5 Measu e iame e o / -I w + C U
ix LIST OF FIGURES
Figure Page
I.1 (a)Normal arteriovenous anatomy, (b) AVM, (c) advanced AVM with down stream aneurysm 4
I.2 I,3-bis (2-chloroethyl)-1-nitrosoureal 1I
I.3 Reservoir systems (a) implantable/oral, (b) transdermal 17
1.4 Environmentally responsive systems (a) reservoir, (b) matrix in swelling- controlled release systems 18
1.5 Basic changes in polymer structure for environmentally sensitive systems due to swelling 20
I.6 (a) Bulk erosion drug delivery, (b) surface degradation for drug release .... 22
I.7 Microspheres of 60 lactide:glycolide PLGA 23
1.8 75:25 lactide:glycolide PLGA microparticles by bulk hydrolysis 23
I.9 (a) surface erosion of polyorthoester rods after 9weeks and (b) l6weeks ..... 23
1.I0 Ring opening polymerization of glycolide dimer to polyglycolic acid 24
1.11 Ring opening polymerization of lactide to polylactides 25
I.12 Degradation and metabolization of PLGA 28
2.1 (a) Fluid jet, (b) Splaying, (c) Whipping motion 30
2.2 Electrospinning setup 3I
5.1 Branching morphology observed in glove samples using OM, magnification of 50 46
5.2 (a) 80/20-10wt% 11/19/03 2 gauge 48
5.2 (b) 80/20-6wt% 1I/20/03 22 gauge 49
5.2 (c) 80/20-6wt% II/20/03 22 gauge 49
5.2 (d) 80/20-10wt% 11/25/03 20 gauge 50 IS O IGU ES (C nt n d
r
5 (e / -1 w 1I/ 5/ 3 gauge 5
5 ( / -1 w 1I/ 5/ 3 gauge 51
5 (g / -I w 11/ 5/ 3 gauge 51
5 ( / -1 w 1I/ 5/ 3 gauge 5
5 (i 5/15- w 1 / 1/ 3 gauge 5
5 ( 5/15- w 1 / 1/ 3 gauge 53
5 (k 5/I5- w 1 / I/ 3 gauge 53
5 (1 5/I5- w 1 / 1/ 3 gauge 5
5 (m 5/15-1 w 1 / / 3 gauge 5
5 ( 5/15-1 w 1 / / 3 gauge 55
5 (o 5/15-1 w 1 / / 3 gauge 55
5 ( G o e sam e-mu i 11/15/ 3 5
5 (q G o e sam e-mu i I1/I5/ 3 57
5 ( 5/I5-15w 1 /I7/ 3 gauge 57
5 (s 5/I5-15w 1 /I7/ 3 gauge 5
5 ( / -1 w + C U I /I / 3 gauge 5
5 (u / -1 w + C U 1 /I / 3 gauge 59
5 ( / -I w + C U 1 /1 / 3 gauge 59
5 3 (a GA o 1 w a o i e wi —3 w mass oss 3
5 3 ( GA o 1 w a o i e wi —3 w mass oss
5 (a SC o / GA i s ea cyc e
x LIST OF FIGURES (Continued)
Figure Page
5 ( SC o / GA i s ea cyc e 7
5 (c SC o / GA i s ea cyc e
5 ( SC o / GA i s ea cyc e 7
5 (e SC o / GA i s ea cyc e 71
5 ( SC o / GA i s ea cyc e 7
1 I c ease is a ce iame e ec eases om 5um o 333 m 73
ii C A E
I O UC IO
. Obj t v
The objective for this thesis research is to use the electrospinning process in order to create a D, L poly-(lactide-poly-glycolide) (DLPLGA) drug delivery system, using the drug 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), to target intracranial vascular malformations and tumors. By creating such a system to the nanoscale, delivery of the system can be done by a guided catheter (endovascular techniques) and decreases the invasiveness of the treatment procedure in comparison to current practice regimens.
By using the electrospinning technique, a non-woven sheet of nanofibers is created by which porosity, surface area, fineness, uniformity of fiber diameter, and pattern thickness of the sheet can be manipulated. The chemotherapeutic agent, BCNU, is known to alkylate the DNA of tumor cells causing apoptosis. To conduct the research,
DLPLGA, a widely used polymer for drug release with well-characterized degradation properties in concentration ratios of 85/15 (high inherent viscosity [IV], GMP), 80/20
(high IV, not GMP), and 75/25 (high IV, GMP) was used. All three ratios were tested in order to obtain the ideal method for drug delivery. Each of the ratios had different degradation rates (different release profiles), therefore met different standards for the system.
In order to define the ideal system, the electrospinning process allows for
DLPLGA nanoscale fibers to be conjoined with BCNU and deliver the drug through a
steady degradation rate over a targeted period of time. The release rate of the drug is dependent upon the degradation rate, which is based upon the characteristics of the
2 polymer. By doing so, it is anticipated that the drug would be packaged in its most stable state and concentrated form for immediate release. The solvent, Tetrahydrofuran (THF), would allow for the insertion of the delivery of this system through a guided catheter, performed by an endovascular surgeon. The drug would be released by oxidation (by means of hydrolysis) of the nanopolymer fibers at target site, thus providing high concentrations of drug to the tumor bed while potentially greatly limiting any systemic side effects.
The research done in this thesis is an initial step in creating a more complex drug delivery system. An electrospun sheet will most certainly not be the final physical form for the delivery, but it is sufficient for the initial characterization of the system for proof of concept.
.2 r nd Inf r t n
It is essential to identify the problem and create a solution that has the ability to have variance in drug choice and material choice. In neuroscience, in choosing AVM's and intracranial tumors as the problem, an extensive variety of uses for the drug delivery
system were available. The use of BCNU as the drug of choice was to narrow down a focal point for the drug delivery system model. With BCNU to create and test the model
system, it could then also be determined if other drugs could be used in place of the
BCNU with a set of data for comparison. The sheet formation used in this model is not the ideal morphology, but is compatible for the initial stages of the research. Other electrospun polymer morphology is plausible and should be investigated. 3
1.2.1 Intracranial Central Nervous System Disease
Arteriovenous Malformations (AVMs) are an abnormal collection of blood vessels that affect approximately 250,000 people (one out of I000 people are born with an AVM) in the United States [7]. Histologically, these are lesions (also known as an AVM nidus) that (typically located as a connection between the artery and the vein) provide nutrient and gas exchange to the tissue. The resultant low-resistance, high pressure and high flow state within the nidus creates a shunt. Thus, in an AVM, blood flows from arteries
straight through the shunt and into the venous system [6]. Consequently, much of the blood can be diverted from the surrounding tissue in order to flow through the AVM,
since the AVM provides less resistance to blood flow than the capillary network.
Increased blood flow through the AVM increases the pressure on the vessels, which can result in formation of aneurysms and possible hemorrhage (2- 4% chance of bursting per
year) [Figure 1.1] [7]. In short description, 1 point is given to a small tumor, the size of
approximately 3cm or an eloquent or deep AVM. Non-eloquent and superficial AVM's
are given zero points. Medium size AVM's are approximately 3-6cm with a value of two
points and large size AVM's in reference to greater than 6cms with a value of three
points. 4
(a)
(b) (c)
r . (a) Normal arteriovenous formation, (b) AVM, (c) advanced AVM with down stream aneurysm.
Cerebral AVMs represent about 80% of all intracranial vascular malformations.
Exact pathogenesis is not known though a male preponderance is reported by almost all studies [8]. Incidence of bleeding is about 70% over course of lifetime [8], and is unrelated to stress, trauma, and hypertension. There exist some studies that show the possibility of rupture during the onset of pregnancy (questionable). However, in general, the risk of bleeding is greater in children, but risk percentages decline after the age of 40
[8]. There is some evidence to suggest that because of specific hemodynamic reasons, small AVMs have a higher chance of rupture due to the build up of higher pressure in the feeding artery. 5
In addition to hemorrhage, seizures are a common presentation of AVMs
occurring in about 30% of all cases and are associated with subclinical development in
about 7% of cases with the average age of onset of 25years [8]. Malformations are so complex, because they can affect individuals during the most productive years of their
lives with such devastating consequences, and because current treatment carries
substantial risk, developing a system, which can safely and effectively treat these lesions
is vital. A drug delivery system that is compatible to all and accessible to all areas is
favored.
Primary malignant tumors of the central nervous system, despite years of study,
still carry a mean life expectancy of 13 years. There exists a four-tier system for
assessing tumor activity and progression (as per the world health organization [WHO]):
Grade 4 being rapidly growing and malignant and 1 being the slowest. The area of the brain and the size of the tumor are factors involved in determining the grade level. For
the lowest grade, grade I, treatment can be as simple as surgery. However, for higher-
grade tumors, the solution is not as simple due to the invasion of cancerous cells into
surrounding tissue, which may even be transported by spinal fluid to completely different
regions of the brain, and spinal region.
Each of the grade levels applies to each type of tumor, with slight variations in
description based on the cell characteristics and treatment methods. Astrocytomas are
tumors formed from astrocyte cells, which form part of the brain's supportive (neuroglial)
tissue [9]. Grade 2 has a 34% 5 year survival rate without treatment. With radiation
therapy, survival increases to about 70%. With grade 3, the average survival of patients
is 18months, with diagnosis by surgery or stereotactic biopsy and follow-up with 6 radiation therapy and chemotherapy [9]. In grade 4 patients, the average (mean) survival after diagnosis without treatment is 17weeks. Survival is extended up to 30weeks with biopsy followed by radiation therapy and 37weeks with surgical removal of most of the tumor tissue combined with radiation therapy. The best treatment involves stereotactic volumetric resection of the tumor tissue component followed by radiation therapy, for a patient longevity of approximately 51 weeks. Glioblastoma Multiforme (grade 4 astrocytoma) makes up 25% of all primary brain tumors. This tumor is able to infiltrate throughout the entire brain and is marked by areas of necrosis (dead tumor cells) [9].
Metastatic tumors are caused by cells from other tumors in the body transported through the blood stream to the brain. The brain tissue around the tumor begins to swell
(edema), causing focal neurological deficits, lethargy, and in extreme cases coma and death. Oligodendrogliomas originate from oligodendroglial cells that form myelin, the fatty substance that surrounds the axons of nerve cells and provides the insulation, which makes nerve cell electrical transmission faster and more efficient [9]. Tumor growth depends on the rate of mitosis and rate of apoptosis. When the mitotic rate of the oligodendroglial cells has exceeded the mitotic rate of the other cells, therefore becoming the dominant cell type, the degree of classification time changes from 6months to 30 years [9]. Most glial tumors have two growth patterns: solid tumor tissue and infiltrating tumor cells, which are judged by progressive enlargement on a computerized tomography
(CT) or magnetic resonance imaging (MRI). Solid tumor tissue is characterized by the increase in mass from the production of new blood vessels from those that supply the brain with oxygen, glucose and other nutrients. The new blood vessels function to supply the growing tumor cells with oxygen, glucose and other nutrients, directly infiltrating the source of nutrients that should supply the brain. Low grade (low blood supply needed) tumors may not be apparent on a CT or MRI due to lack of contrast enhancement
(anaplastic or malignant require large blood supply) and may be removed without neurological deficit (solid tumor tissue either displaces or replaces brain substance) depending on the lesion's location [9].
Isolated tumor cells can move between the fibers of the brain, with the entire tumor consisting of these cells which reside in "sick" but still functioning brain tissue, which makes removal difficult [9]. Most low-grade infiltrating tumor cells are visible as a hypodensity on a CT and increased Ti and T2 signal on an MRI because the tumor cells tend to draw water into the infiltrated brain tissue [9]. If isolated tumor cells are not great enough in number to change the local osmolality and thus increase the amount of water surrounding them, the CT and M I will show no changes and appear normal in the area [9]. If some tumor cells still reside in the tissue after treatment, it is guaranteed that the tumor will return. These types of tumors are more frequent in young and middle-aged adults.
.2.2 C rr nt r t nt M th d
To locate problematic areas, computerized tomography (CT's), magnetic resonance
imaging (MRI's), and Angiogramss (x-ray movies of the blood flowing through the blood
vessels, made by injecting contrast into the arteries going into the head and taking a series
of x-ray films) are used. However, not all of these methods are completely accurate. As
mentioned in section 1.2.1 Intracranial Central Nervous System Disease, there are a
variety of lesions whose behavior is not always predictable or traceable. If the cells do 8 not produce enough of a difference in osmolality compared to normal conditions, a CT or
MRI may not detect the malformation.
Embolization is a method of plugging the blood vessels of the AVM, by an x-ray guided catheter from the femoral artery in the leg up into the area to be treated [6]. The
AVM's feeder vessels are then sealed off from within using an epoxy applied by an angiographic catheter [7]. Embolization is usually done in preparation for surgery to reduce the blood flow within the AVM or is combined with radiation therapy to obliterate the AVM. The benefits of this method are that: (1) it does not require a craniotomy; (2) eloquent deep areas can be treated by doing small areas at a time and allowing the
surrounding brain to recover; and (3) testing of the importance of the area can be done by injection of medication into the area before permanent treatment is done [6]. However, it requires multiple treatments, multiple entries into the brain, the use of new materials under "Investigation" (not yet approved by the FDA or not GMP regulated), and is used as an adjunct to another method (often combined with surgery or radiosurgery) [6].
Radiation therapy is a noninvasive procedure that uses ionizing radiation to damage the DNA in all cells (normal cells can repair damage but tumor cells cannot).
Irradiated tissue temporarily swells, that there exists a maximum volume above which the amount of swelling can cause damage. Therefore to minimize the dose given, the radiation beam is directed from several angles toward the site by a linear accelerator.
The efficacy of radiation treatment is limited by the size of the lesion, with the limit at no greater than 3cm due to drastic reduction in cure rate, with a concomitant increase in complication rate. There are different techniques being developed for radiation therapy.
Stereotactic Brachytherapy is an invasive method that uses multiple sources placed within tumor tissue to provide a dose field, with 40% of patients requiring an operation
afterwards [9]. Boron Neutron Capture Therapy (BNCT) uses the theories of atomic physics in which a boron compound is taken up by tumor cells, neutrons are absorbed
into the Boron, forming gamma irradiation and an alpha particle which kills the tumor
cell [9]. This method is usually used for tumors and not AVMs.
Surgery is an invasive procedure that involves the excision of some or the entire
lesion, with preservation of normal brain tissue. There are three steps to the procedure:
(1) establish tumor cell type and grade, (2) relieve internal pressure, and (3) reduce the
tumor burden. A craniotomy is where a portion of the skull is temporarily removed,
allowing the surgeon access to the lesion. Stereotactic procedures precisely localize areas
inside the head for biopsy, such that a stereotactic biopsy (probe directed by stereotactic
frame to tumor) is performed. Another similar method, the computer-assisted volumetric
stereotaxis gathers, stores and reformats imaging-derived, 3-D volumetric information
defining an intracranial lesion with respect to the surgical field in order to plan and
simulate the surgical procedure beforehand [9]. Volumetric stereotaxis can minimize the
incision and subsequent injury to brain tissue, thus maximizing tumor removal and
reducing postoperative complications and neurological morbidity [9]. Stereotactic
radiosurgery claims a success rate of 80% after two years of follow-up, depending on the
histologic grade of the lesion [8]. As efficient as all these methods are, surgery still is an
invasive process that involves the risk of bleeding and infection, a long recuperation time,
and is dependent on how accessible (how deep into the brain tissue) the lesion is.
Chemotherapy proves to be a promising area for advancement in approaching the
problem from a different perspective. Most agents are incorporated into and alkylate the 1
DNA of rapidly dividing tumor cells like BCNU, PCNU, Procarbazine and Carboplatin.
Vincristine poisons the mitotic process and VP-16 (Etoposide is a semisynthetic derivative of podophyllotoxin used in the treatment of certain neoplastic diseases) incorporated in tumor cell wall proteins inhibits the production of microtubules.
Topotecan enhances the effect of radiation therapy by intravenously affecting rapidly dividing tumor cells and normal cells from bone marrow and bowel. Most of these agents may result in the reduction of white blood cells (causing severe, potentially life- threatening infections), red blood cells (causing anemia), and platelets (thrombocytopenia
- resulting in blood clotting disorders and bleeding). However they are the only agents that can overcome the problems with glial primary brain tumors. Many tumor cells infiltrate surrounding brain tissue where the blood-brain barrier (which excludes large molecules) is intact. These agents can reach cells in the major mass of the tumor, which is supported by leaky tumor blood vessels, via these blood vessels. The blood-brain barrier protects tumor cells residing within intact brain tissue surrounding the tumor, but can be disrupted medically by chemotherapy.
Guilford pharmaceuticals markets polyanhydride Gliadel wafers for treating a type of brain cancer called glioblastoma multiforme. Wafers consist of new polymers impregnated with carmustine (BCNU), a toxic antitumor medication and are implanted at the tumor site, release the drug locally where it is needed while reducing systemic side effects such as liver or kidney damage. However, the wafer does have other side affects due to the drug delivery system and the means of implantation. In clinical trials, I9% of patients reported new or worsened seizure symptoms and 14% experienced healing abnormalities (such as cerebral fluid leaks, subdural fluid collections, wound 11
eak ow O e a e se eac io s i c u e i ac a ia i ec io s ai i ack a c es y e e sio ia ea e e as es a u i a y ac i ec io s Com i a io o
ea me s is e es me o i cu i g ai umo s a A Ms ey ca e ai o e o
e s eci ic A M y e o umo y e a a ie co si e a io s u equi es a i io a s e s ime a coo i a io [ ]
1.2.3 Pharmacokinetics of BCNU
C U [1 3- is ( -c o oe y -I- i osou ea] a so k ow as ca mus i e as e s uc u a o mu a ou i igu e 1
O
C CH2-CH2-NCNHCH2-CH3-CI
NO
Figure 1.2 [1,3-bis ( -c o oe y -1- i osou ea]
e c emica ac i i y o C U is o a ky a e e A a A o ce s I as
ee k ow o eg a e o s o a eous y a me a o ica y e A c oss- i ks
y o esi e o e i e om c o oe y ca o ium io [ ] ase o i a e ous s u ies e a e age e mi a a - i e ( ase o osage a ge om 3 o 17 mg/m was mi u es wi a c ea a ce o 5 m /mi /kg a a s ea y-s a e o ume o
is i u io o 3 5 /kg [ ] In a mg/m i a e ous osage -7 was e c e e oug u i e a -1 e i e as ca o io i e o e a 9 ou ime s a 2
e emai i g -3 as o ee accou e o ye e eg a a io a e occu s a suc a ig a e a wi i 15mi u es o i ec io o i ac ug is e ec a e S u ies
a e s ow a e eac i e a u e o e ug is so e e si e a i ca e ca ie y ce e a oo ow o ce e os i a ui ow a o e come e oo - ai a ie wi i e co i es o ai issue a is a ce away om e im a a io si e o e e ice
e oo - ai a ie is o e come ecause o e com ou s ig i i so u i i y a e e a i e ack o io i a io a ysio ogica ue o i s o ic a u e
C U may a so i i i se e a key e yma ic ocesses y ca amoy a io o ami o aci s i o ei s o g- e m usage o a i osou eas o uc is associa e wi
e e o me o seco a y ma ig a cies I as a so ee s ow o e em yo o ic i
a s a a i s a i i ec e i a i aa e ia i aca o i ou e associa e wi ocu a
o ici y I a s u y o e o ma e a s gi e e osage a o e o uma s e i i y was im ai e a ca ci oge ic a mu age ic e ec s we e o e I a e ous y a mi is e e ma y o e si e a ec s a e co cu e u o e i e y ese a e u mo a y
o ici y (suc as i osis gas oi es i a o ici y e a o ici y e o o ici y a ski
y e igme a io u o co ac
e y o m o e ac i e age ca e s a e o u o wo yea s i em e a u e o °C - °C (3 ° - ° a w e sus e e i i ue eig ou s a oom
em e a u e away om su ig I as a ow me i g oi a 3 5°C o 3 °C ( 9° -
9 ° a ca e o ess y ique y ( om y o m w e em e a u es c a ge 13
1.2.4 Biodegradable Polymers
The first FDA-cleared PLGA product was the Lupron Depot drug-delivery system (TAP
Pharmaceutical Products Inc.; Lake Forest, IL), a controlled release device for the treatment of advanced prostate cancer that used biodegradable microspheres of 75:25 lactide/glycolide to administer leuprolide acetate over periods as long as four months
(replacing daily injections) [I3]. "Another drug-delivery device, the Gliadel Wafer
(Guilford Pharmaceuticals Inc.; Baltimore, MD), is used to prolong the life of patients suffering from a particularly deadly form of brain cancer, glioblastoma multiforme. In this case, dime sized wafers of a biodegradable polyanhydride copolymer-poly [bis (p- carboxyphenoxy) propane: sebacic acid] in a 20:80 molar ratio - are implanted directly into the brain to deliver a powerful chemotherapeutic agent (BCNU) that has deleterious side affects when administered systemically [I3]. Release of the drug delivery in the implantable wafers was stimulated by the presence of poly (N-vinylpyrrolidone) (PVP) or sodium chloride (NaC1).
However, using different biomaterials raises considerations on biocompatibility, the reaction of the patient to the material, the effectiveness of the drug delivery system, and the cost [Appendix B]. For the purpose of this research, the amorphous form of
Polyglycolic acid/Polylactic acid copolymer (PGLA or PLAGA) (some ratio of it discussed later) was chosen for the base material. This biodegradable synthetic polymer is amorphous with a weight of 40-100kD. It has a decomposition of 100% in 60-90 days
(observed in rat stem cells). A biodegradable polymer is optimal for drug delivery systems by controlled processing so that optimal release of kinetics of the drug or active agent is achieved. Polymer degradation is accelerated by greater hydrophilicity in the 14
ack o e o e g ou s g ea e eac i i y amo g y o y ic g ou s i e ack o e ess
c ys a i i y g ea e o osi y a sma e i is e e ice si e [13]
W e co si e i g a io eg a a e o yme o e as o i k a ou e
ysio ogic e ec s e mec a ica o c emica ocess a ei o uc s may a e o
e su ou i g a ea I a s u y o e i A a a Geo gia i o yme eg a a io
a ec e ce g ow e a a s owe a i ec a i io o ei e g yco ic o ac ic
aci s o co o ce cu u es wi ou AGA esu e i o o io a cy o o ici y [1 ]
e me a o ic eak ow o uc s om AGA sig i ica y owe e e o e e i o me a a sus ai e ce ia i i y o AGA mem a e is a ia y e e e o e oca es ecia y i co o e imi e in vitro cu u es [1 ]
is ossi y is e e icia si ce e goa is o e e ce g ow S i cau io mus e use i e e i g amage o ia e o -ca ce ous ce s I a i io e ow o e oo s ou us e esi ua com o e s ou o e sys em e is o a y
o a issue ue o e e eme u e i g ca aci y o oo w ic wi co i ua y was away a y e ease o uc s om e eg a a io o e GA o yme e
i e e ce may o y e a co ce w e ea i g wi e ug e i e y e ac ua ug
e o ma ce a a y co i io s e a ie may a e a may e a ec e y e c a ge
G A is com ise o A a GA i es ec i e a ios o co i u e o e mec a ica oug ess a s e g ou i ei e e c ys a i e o e semic ys a i e
o m use o io ogica a ica io [A e i A] o yme s a e e y ow
o y is e i y i e a ios (PDI) a c a ac e i e e mai e a ce o e mec a ica a s uc u a s e g [1 ]
A e o yme s a e e y ow o y is e i y i e a ios ( I o e am e
e I a io o A is a ou 1 -1 9 i o e o mai ai mec a ica a s uc u a co sis e cy U i i i g i g-o e i g o yme i a io com i es e mos commo me o o comme cia o uc io o A a GA wi a i se io mec a ism usi g a me a o i e [1 ] A mo e amo ous o m o e o yme ca e use o ug e i e y
e ices w i e e c ys a i e o m is goo o ui i g sca o i g a o e
io eg a a e s uc u es GA o e am e is com e e y amo ous so e e o e i is use o y i ug e i e y e ices e use o ce ai ugs o e am e is o i i e
y e e a i e y ig em e a u es use i co s uc i g ese o yme s A o e
aw ack is i e co o e e ease o ugs u k e osio as a somew a i co sis e
e ease o ug e e i g o e amou o ug oa e o o e o yme i s
y o i ic o y o o ic o e ies e i i ia a e o e ease ca a y [1 ]
.2. K n t f r l
Co o e ug e i e y is i ec y e e e u o e a u e o e io eg a a e
o yme (i eg a a io is e co o o e ease a e e ac o s a a ec e ease a e a e c emica s uc u e c emica com osi io ocessi g co i io s mo o ogy a si e o im a a io C emica s uc u e i c u es mo ecu a -weig is i u io s a e a ysica ac o s (s a e a si e c a ges a ia io s o i usio coe icie s mec a ica s esses s ess- a so e -i uce c acki g C emica com osi io i o es e ese ce o io ic g ou s mo ecu a weig e ese ce o ow-mo ecu a - weig com ou s a so e a a so e com ou s (wa e i i s io s
ysicoc emica ac o s (io e c a ge io ic s e g a mec a ism o y o ysis 1
(e ymes e sus wa e [ ] ocessi g co i io s wi s o age is o y s e i i a io
ocesses a a ea i g a e a a ec o e i a o e a u c io a i y o e e ease sys em is i u io o e ea u i s i mu ime s e ese ce o u e ec e u i s o c ai e ec s a co igu a io s uc u e a a u e e ca ego y o mo o ogy
(amo ous/semic ys a i e mic os uc u es esi ua s esses [ ]
e e ease o e ac i e age may e co s a o e a o g e io i may e cyc ic o e a o g e io o e e i o me o o e e e a e e s may igge i i o e o mai ai e co ec amou o osage ( e e i g o e - a u e osi g [ ]
e e a e ee ima y mec a isms o ug e ease
(I i usio ; ( eg a a io ; (3 swe i g o owe y i usio I some ug e i e y sys ems a com i a io o e mec a isms was use i usio ca occu o a mac osco ic sca e as oug o es i e o yme ma i o o a mo ecu a e e y
assi g e wee o yme c ai s w e e i o cases e mo o ogy o e o yme is
e co o e - e ease e ice [ ] i usio a so ca occu w e e ug asses om
e o yme ma i ( omoge ous sys em o me om e u io o o yme a ac i e age i o e e e a e i o me As e e ease co i ues i s a e o ma y ec eases wi is y e o sys em si ce e ac i e age as a og essi e y o ge is a ce o
a e a e e o e equi es a o ge i usio ime o e ease [ ]
ese oi sys ems a e esig e o use e me o o i usio y e c osi g a ac i e age (so i ug i u e so u io o ig y co ce a e ug so u io wi
o yme ma i wi a ma e ia a ac s as a mem a e aci i a i g e i i g o e e i g mo ecu es a a co sis e a e o e ese oi sys ems s ow i igu es I a a e
ug e i e y a e ca emai ai y co s a [ ] e mem a e o e ese oi ca 17
a e a u i o m o yme coa i g o ei e o e si e o o si es e e i g o w a e s imu us o e ac i e age is e sys em s ow i igu e 1 3(a is e ese a i e o a im a a e o o a ese oi e i e y sys em w e eas e sys em s ow i igu e 1 3( i us a es a a s e ma ug e i e y sys em i w ic o y o e si e o e e ice wi ac ua y e e i e i g e ug [ ]
e e i o me e ac i e age is e ease i ca egu a e co o o e ug
e i e y O ce e ac i e age as ee e ease i o e e e a e i o me a a i io a se ies o i usio a a ac i e a s o s e s ca occu a a e a i ec
e a io s i wi e i o me a ac o s
(
igu e 1 3 ese oi sys ems (a im a a e/o a ( a s e ma 18
In these systems, the combinations of polymer matrices and bioactive agents
chosen must allow for the drug to diffuse through the pores or macromolecular structure
of the polymer upon introduction of the delivery system into the biological environment
without inducing any change in the polymer itself [20]. Environmentally responsive
systems [Appendix K] when in contact with a particular biological environment, release
agent or agents in a stable way based on the kinetics of osmolality. An example of this
type of mechanism is a swelling-controlled release system. These systems operate by
absorbing water or other body fluids, which induces swelling, causing the dilution of the
active agent and the enlargement in the size of the polymer mesh. Diffusion then takes
over as the means of drug release. Figures 1.4(a) and 1.4(b) are examples of these types
of devices for reservoir and matrix systems, respectively [20]. The theory behind this
particular system is based on hydrogels, polymers that swell without dissolving in
aqueous solutions. At equilibrium, hydrogels comprise 60-90% fluid and 10-30%
polymer.
(3
Figurel.4 Environmentally responsive systems (a) reservoir, (b) matrix in swelling-controlled release systems.
The onset of swelling is not just dependent upon contact with a particular environment, but also with changes in the environment. Changes in the environment include temperature, pH, ionic strength, introduction of ionic groups into the environment, ion exchange, variations of diffusion coefficients, and enzymes or other biological elements that can induce shrinkage or swelling. Hydrophilic excipients can be used to accelerate the release of drugs, though they may also increase the burst effect
(release of active agent at one time) [18].
The simplest environmental design relies on the difference in pH values, where at high pH values, the system swells and at low values it collapses. The swelling, induction of high pH values triggers the drug release and low pH values arrests release. A number of these environmentally sensitive or "intelligent" hydrogel materials are listed in
[Appendix K]. For most of these polymers, the structural changes are reversible and repeatable upon additional changes in the external environment, as illustrated in Figure
I.5 [20]. The drug release mechanism of the system in Figure 1.5 is based upon the
swelling of the polymer. Swelling is triggered by an increase in pH value and is ideal for oral delivery where release occurs in the upper small intestine where the pH values are high and not in the stomach where pH values are low. 20
Figure 1.5 asic c a ges i o yme s uc u e o e i o me a y se si i e sys ems ue o swe i g
Co a y o e esig o e swe i g sys ems a e e io eg a a e sys ems
io eg a a e sys ems ma i u a e a u a io ogica ocesses o eg a e e o yme
e e o e e easi g e ug a was a e i o o e o yme I suc sys ems e c emica s uc u e o e sys em c a ges a e e ease a e is o e go e e y e
eg a a io a e i some e a io s i Mos io eg a a e o yme s a e esig e o
eg a e ia e y o ysis o e u s a e o yme c ai s as soo as ey come i o co ac wi wa e I e sim es mec a ism o c emica y o ysis wa e e e a es
e u k e e e ia y a acki g e c emica o s i e amo ous ase a co e i g o g o yme c ai s i o s o e wa e -so u e io ogica y com a i e com ou s o some o yme ma e ia s suc as o y ac i es o yg yco i es a ei co o yme s e o yme s wi eak ow o ac ic aci a g yco ic aci e e e
K e s cyc e a e u e oke ow i o ca o io i e a wa e a e c e e ou 2 o e o y y o ma io ogica ocesses Si ce eg a a io i i ia y occu s i e amo ous ase e e uc io i mo ecu a weig oes o a ec ysica o e ies o u c io a i y o e e ice
e e ice is e oge e y e c ys a i e egio s a emai s i ac u i e
eg a a io eac es a c i ica oi omo o yme s ( o yg yco ic aci a o y ac ic aci a e c ys a i e e se y acke a a e o i e y o y ic a ack u ike co o yme s w ic a e amo ous o amo ous o yme s a y o o ic aye (a co o yme o ac i e g ac i e a ca cium s ea a e o e su ace ca e ge e a e
o mi g a a so a e a e e o - aki g u ica w ic e e s wa e (mo ecu es ca o eac c ai segme s i amo ous a eas o i i ia e y o ysis s ows a so io a im o es e e e io o e si e s e g [17]
O ce e e ice is oke ow e agme s a e u e e uce o sim e com ou s ue o e yma ic a me a o ic a ec s W e suc a ac io occu s w e e
e e is a a i oss o o yme mass ( e a e a w ic wa e e e a es e e ice e cee s a a w ic e o yme is co e e i o wa e -so u e ma e ia s i is ca e
u k e osio [1 ] oug u k e osio e o yme eg a es i a ai y u i o m ma e oug ou e ma i o y su ace eg a a io as i suc ma e ia s as
o ya y i es a o yo oes e s I su ace eg a a io e e ease a e is
o o io a o e su ace a ea o e ug e i e y sys em esu i g i a s owe a e o co e sio o e o yme i o wa e -so u e ma e ia s A y o o ic o yme w ose c emica o s a e ig y susce i e o y o ysis ca e e ie ce a o m o su ace
eg a a io u is o e e e e o as ioe osio [1 ] igu e I (a i us a es u k e osio a igu e 1 ( s ows su ace eg a a io
igu e 1 (a u k e osio ug e i e y ( su ace eg a a io o ug e ease
Mic o a ic es a e a e am e o u k e osio sys ems use mos commo y i o a e i e y sys ems a su cu a eous y i ec e e i e y sys ems Mic o a ic es o
o y ( ac i e-co-g yco i e ( GA ca e e a e i a ai y u i o m ma e o o i e esse ia y o o ous mic os e es [ igu e 1 7] a e uce o agme s y u k
y o ysis [ igu e 1 ] (e am e a e o a 75 5 ac i e g yco i e GA mic o a ic e a e 133 ays o eg a a io i wa e [ ]
A a ysis o o yo oes e (su ace-e o i g o yme s o s a e 9 a 1 weeks o im a a io i a i s s ows sig i ica su ace eg a a io u e co e o e ug
e i e y sys em emai s i ac [ igu e 1 9] [ ] 2
r . Mic os e es o ac i e g yco i e GA
r .8 75 5 ac i e g yco i e GA mic o a ic es y u k y o ysis
(a (
r . (a su ace e osio o o yo oes e o s a e 9weeks a ( 1 weeks 24
.2.6 GA Ch r t r t
Poly-glycolic acid (PGA) [Figure 1.I0] is the simplest linear aliphatic polyester, with a
melting point of 220-225°C and a glass-transition temperature of 35-40°C due to its high
crystallinity (45-55% crystalline). Such high crystallinity prevents solubility in most
organic solvents with the exception of highly fluorinated organics such as
hexafluoroisopropanol. Ring-opening polymerization yields high-molecular-weight
materials, with approximately I-3% residual monomer present and dimerization of
glycolic acid results in glycolide monomers. Unlike the slow absorption of Poly-lactic
aci ( A GA is a so e wi i a ew mo s os im a a io ue o g ea e
y o y ic susce i i i y [15]
11- - - I I 1 5 4 3
G yco i e - ime o o yg yc o ic Ac i G yco ic aci
r . 0 i g o e i g o yme i a io o g yco i e ime o o yg yco ic aci
I i o e e ime s co c u e a e ymes u e (mois u e a ea i g
ea me s a gamma a ia io e a ce eg a a io o GA e e o e ow umi i y
e y e e o i e gas s e i i a io oce u es a mois u e- oo ackagi g a e u i i e
Gamma i a ia io ca e use w e e o se o eg a a io is esi e a a as e a e 25
PLA (poly-lactic acid) [Figure 1.II] is prepared from the cyclic diester of lactic acid
(lactide) by ring opening polymerization. Lactic acid exists as two optical isomers or enantiomers (D and L), the L-enantiomer (crystalline) occurs in nature, and a D, L racemic mixture (LPLA, a semicrystalline polymer) can be made by synthetic preparation
of lactic acid.
n
ac i e o y ac i e ( i e o ac ic aci
Figure 1.11 Ring opening polymerization of lactide to polylactides.
Poly-L-lactide is about 37% crystalline with a melting point of 175-178°C and a
glass transition temperature of 60-65°C [I7]. Electrospun fibers from L-polylactide (mp.
I70 C) have high crystallinity, whereas poly DL-lactide electrospun fibers are amorphous
(no reported melting point [mp]). Poly L-lactide is more resistant to hydrolytic
degradation than the amorphous DL form, due to its crystalline nature, which exhibits
high tensile strength and low elongation, resulting in a high modulus (more suitable for
load bearing devices). DLPLA is an amorphous polymer exhibiting a random distribution
of both isomeric forms of lactic acid, is unable to arrange into an organized crystalline
structure, and has lower tensile strength, higher elongation, and faster degradation time
(ideal for drug delivery systems). 26
Carboxyl-ended PLGA polymers degrade slower than hydrophobic end-capped
PLGA polymers. Polylactides are more hydrophobic; therefore take a longer time to
degrade, unless plasticized with triethyl citrate (producing a less crystalline, less tensile,
faster degrading material [17]). Time required for poly-L-lactide implants to be absorbed
depends on polymer quality; processing conditions, implant site, and physical dimensions
of the implant [I5]. In vivo studies in rats showed an absorption time of about I.5 years
for 50-90 mg samples of radiolabelled poly-DL-lactide implanted in the abdominal wall
[15]. In the radiolabelled implants, metabolism of the polylactides resulted in excretion
primarily via respiration (CO2) and exposure to gamma radiation showed a decrease in
molecular weight (MW). The degradation time of LPLA takes more than two years to be
completely absorbed, much slower than DLPLA. Copolymers of L-lactide and DL-
lactide have been prepared to disrupt the crystallinity of L-lactide and accelerate the
degradation process [17]. The rate of degradation of lactide based polymers and in
general all hydrophobic degradable polymers, depends on chemical composition,
crystallinity, and hydrophilicity. Degradation by chemical composition depends on the
rate of degradation of the bonds present (Anhydrides faster than esters, faster than
amides). The higher the crystallinity and hydrophobic nature (versus hydrophilic), the
slower the degradation rate. Schwendeman and Zhu (Zhu and Schwendeman, 199 et al.,
2000) have shown that by incorporating basic salts as excipient polymeric microspheres, the stability of the incorporated protein improved, with the side effect that these basic
salts slowed degradation time.
Amorphous copolymers have a compositional range between 25-70 mole percent glycolide, where pure polyglycolide is about 50% crystalline and pure poly-L-lactide is 2 about 37% crystalline. There is a disruption of the regularity of the polymer chain by the other monomer in a racemic mixture. A copolymer of 50% glycolide and 50% DL- lactide degrades faster (in 50-60 days) than either polymer independently, or copolymer with differential percentages of either polymer. A copolymer of 90% glycolide and 10%
L-lactide was developed by Ethicon under the trade name Vicryl, which absorbs within 3-
4 months but has a slightly longer strength-retention time [17]. In general, the 65:35,
77:35, and 88:15 D, L-lactide/glycolides have progressively longer in vivo lifetimes, with the 88:15 lasting about I50 days in vivo, whereas poly (D, L-lactide) requires about 12-
16 months to biodegrade completely, and poly (L-lactide), being more crystalline and less hydrophilic, can be found in vivo in about 1-I/2 to 2 years (Cutright et al. I974,
Hausberger & De Luca 1993, Holland et al. 1986, Matsusue et al. I992, Pistner et al.
1993, Yamaguchi & Anderson I993) [19]. Factors that affect the performance of biodegradable polymers (in general) are hydrophilicity, crystallinity, melt and glass- transition temperatures, molecular weight, molecular-weight distribution, end groups, sequence distribution (random versus blocky), and presence of residual monomer or additives. The most common functional groups with these characteristics are esters, anhydrides, orthoesters, and amides. A more hydrophilic backbone, less crystallinity, more porosity, more hydrophilic endgroups, more reactive hydrolytic groups in the backbone, and smaller device size are factors that reduce degradation (accelerates the action of) time. 28
0 H 0 O 1 I I I ( ( " I (C _ )2 + -+ CI _ C _ C _ O + _ C _ C _ O
Figure 1.12 eg a a io a me a o i a io o GA
Me iso o yme s ( o yme s use i is esea c A e i C E G H, I, J)
a e io eg a a e o yes e s a u e go eso io o ysio ogica co i io s a
a yi g a es e e i g o c emica c a ac e is ics o e o yme a a i u es o e
e ice eg a a io occu s ia y o ysis o es e i kages o owe y g a ua e osio
o e e ice ike ei ge e a cou e a s e i a o uc s o G eg a a io a e
ac ic aci a g yco ic aci wa e so u e o - o ic o uc s o o ma me a o ism
a a e ei e o u e me a o i e o ca o io i e a wa e [ igu e 1 1 ] CHAPTER 2
ELECTROSPINNING TECHNIQUE
Electrospinning is the process of charging a drop of polymer in solution with tens of thousands of volts undergoing metamorphosis from a Taylor cone into strands of polymer on the nanoscale size (by spraying). Electrostatic charging of the fluid at the tip of the nozzle results in the formation of a Taylor cone, in which multiple filaments are ejected to produce nonwoven materials that have a porosity factor, high surface area, and fineness and uniformity. The filaments are the result of the jet accelerating and thinning in the electric field, a process known as "splaying." Studies suggest that the most important element operative during electrospinning is the rapid growth of a non- axisymmetric, or "whipping," instability that causes bending and stretching of the jet
[Figure 2.1(a)] [2]. The unstable region of the jet [Fig. 2.I (b)] viewed at exposure times down to the millisecond, has the appearance of an "inverted cone," while in [Fig 2.1(c)], using high-speed photography, a single, rapidly whipping jet was observed by Shin et al.
The whipping frequency is so fast that the jet appears to be splitting into multiple filaments [2].
It was demonstrated by Hohman et al. that there are three different modes which are unstable: (I) the Rayleigh mode, which is the axisymmetric extension of the classical
Rayleigh instability when electrical effects are important: (2) the axisymmetric conducting mode; and (3) the whipping conducting mode. (The latter are dubbed
"conducting modes" because they only exist when the conductivity of the fluid is finite
[3].) Hohman et al. demonstrated that the dominant instability strongly depends on the
29 3
fluid parameters of the jet (viscosity, dielectric constant, and conductivity) and also the
static charge density on the jet.
‘ ig is
(
r 2. (a) Fluid jet, (b) Splaying, (c) Whipping motion.
The actual process of electrospinning involves applying a high voltage to a syringe (or some form of conduit) and pumping a polymer solution through it at a steady rate. Nanofibers of polymer are collected as a nonwoven sheet on a grounded plate below the capillary [Figure 2.2].
In the absence of an electric field, the fluid forms a drop at the exit of the conduit; its size is determined by surface tension, distance between conduit and grounded plate, and pumping rate. When an electric field is applied, a charge is introduced to the fluid that quickly relaxes the fluid surface, creating a tangential stress, resulting in the deformation of the droplet into a conical shape (Taylor cone) [5]
Polymer solution Jet
Pipette ge Metering Pump Taylor cone
High Voltage Collector screen Supply (Rotating or Stationary)
r 2.2 Electrospinning setup.
Hohman et al. demonstrated that when the electric field exceeds the critical value needed to overcome the surface tension, the apex of the cone ejects a fluid jet, where at low viscosity, the jet breaks up into droplets, while at higher viscosity the jet forms a continuous, small-diameter filament. Droplet formation depends on viscosity, therefore the solution or melt viscosity is shown to affect both the processing window for electrospinning and the diameter of the fiber [4]. Electric field parameters also affect fiber morphology.
Rutledge and Shin discovered that the small-diameter fibers generated when the filament became unstable and split into smaller filaments (splaying), was actually the main filament whipping around, stretching into a single long fiber. They demonstrated this concept with poly-ethylene oxide (PEO) solutions of varying concentrations (i.e. viscosity) in water, with KBr to adjust the fluid conductivity. The results of their experimental analysis was that the solution flow rate and the electric field strength were the key operating parameters determining the jet's stability and that the measured current 32 of the jet provided information about the surface charge density on the jet [5]. Larrado and Manley determined that doubling the applied electric field decreased the fiber diameter by roughly half. However, Baumgarten showed that the diameter of the jet reached a minimum after an initial increase in field strength and then became much larger with increasing fields, an effect caused by the pumping rate [4]. Therefore he concluded that increasing the field does increase the electrostatic stresses, creating smaller diameter fibers, but it also draw more material out of the syringe [4].
In general increasing the grounded surface distance, decreases fiber diameter; increasing electric potential (kV), decreases fiber diameter to a critical point; increasing flow rate, increases fiber diameter; increasing concentration wt%, increases fiber diameter, and decreasing the conduit size decreases fiber diameter. CHAPTER 3
MATERIALS
The DLPLGA (D-L-Poly-Lactic-Glycolic-Acid) polymer in ratios of 75/25, 80/20, and
85/15 was obtained from Alkermes in 50gram packages (per ratio). Due to their highly hydrolytic nature, the polymers were kept in airtight bags in the freezer. The samples of
75/25 and 85/15 DLPLGA are GMP approved, but the 80/20 DLPLGA was not.
Obtained from the Medisorb® Polymer Products (division of Alkermes) was the following product information [Table 3.11:
Biodegradation rates depend on: device geometry, porosity, lactide:glycolide ratio, MW/inherent viscosity (IV)/Polymer end groups, and crystalline vs. amorphous character.
Table 3.1 Product Information
85/15 DL 2A 85 Mole % DL Lactide, 15 Mole % Glycolide Acid End Group High IV 0.66-0.80 (DL/G) Degradation range of 5-6months Low IV 0.50-0.65 (DL/G)
75/25 DL COI 75 Mole % DL Lactide, 25 Mole % Glycolide Custom 01 High IV 0.66-0.80 Degradation rate 4-5 months Low IV 0.50-0.65
Standard end group is lauryl ester (capped), polymer identifier marked with either A, M, or C.
A - polymers contain a free carboxyl end group (uncapped)
M - polymers contain a methyl ester end group (capped) which lower inherent viscosity with a higher Tg
33 4
C - o yme s a e cus om o yme s i w ic e e g ou wi a y e e i g o e
a c o e e e C esig a io may a so i c u e o yme a as a a ge e I e e
iscosi y a ge a oes o com y wi i e S a a I e e iscosi y
S eci ica io s e ai e e ow
a ge o yme I e e iscosi y (I S eci ica io a ges
Polymer Ratio Polymer Identifier IV (DL/G) End Group
A ow I 5 - 5 Es e
A ig I - Es e
e mo ecu a weig o e 75/ 5 GA was II3K 1 3 K o e 5/15 sam e a 1 K o e / GA sam e e mo ecu a weig was im o a i e e mi i g ow we e o yme wou go i o so u io a a es ima e o ow e e ec os i i g sam es wou u ou ( iame e si e
O e im o a c a ac e is ics o e o yme s a e ou i A e i C E G I
e so e use was e a y a u a ( ecause i was o e o ew so e s
a cou isso e o e GA a e C U wi ou is u i g e u c io a i y o ei e e o yme o e ug [A e i ]
e ug o c oice was e e e y C a es es igiacomo M om e
eu osu ge y e a me a e U i e si y o Me ici e a e is y o ew e sey
(UM Si ce C U is a commo c emo e a eu ic age i e ea me o
a ious y es o ai umo s sig i ica amou s o a a ega i g i s a macoki e ics a ioa ai a i i y i i e i g e a a io s a e ea i y a ai a e o com a iso maki g
is e i ea c oice o es e a i i y o e i e c emo e a eu ic age s ia a o i e
ec o ogy As me io e i e sec io 1 3 a macoki e ics o C U C U was
e ug o c oice w e G ia e ma ke e ei io eg a a e im a a e wa e s o
e e umo eoccu e ce
e ug was o ai e om e a macy a UM i ow e o m o
isso u io wi e o yme i e so e
o ie a y ame C U
USA a ame S e i e Ca mus i e A a o e c emo e a y age SC
99 (1977
Ac i e Age Ca mus i e
Commo ames C U iC U Ca mus i e
C assi ica io A ky a i g Age i osu ea
A e age a ie osage as a si g e c emo e a eu ic age 15 - mg/m e e y
weeks
o e ia Si e E ec s o e Ma ow Su essio A emia ia ea ow W i e oo
Cou s ow a e e Cou s Ki ey amage u g amage ( u mo a y o ici y
Mou So e ess Swa owi g i icu ies [ 1]
Mus e s o e i e ige a o e o e use a ecom oses a - 9 ° Use wi i
ou s a oom em e a u e a o ec om su ig
A e i a e ous y a mi is e e C U ca c oss e oo ai a ie ue o i s ig
i i so u i i y a ack o io i a io a is a i y eg a e wi o i ac ug
e ec a e a e 5mi u es [ 1] 6
esea c a a ysis s owe a e o ici ies o C U a e ue o me a o i es;
a o ima e y -7 o a o a ose is e c e e oug u i e wi i 9 ou s a
a o ima e y 1 as es i a o y CO e emai i g -3 is u k ow [ I]
e E ec os i i g Assay was o ai e om mu i e sou ces a was
co s uc e y S o a a S a muga Su a am o e Me ica e ice a Co ce a a
e ew e sey I s i u e o ec o ogy ( I e o age sou ce Gamma ig
o age esea c (O mo eac o 5k was use o a y e ec ic ie a
e e o e s
e sy i ge was om is e Scie i ic o si es -1 gages w e e a (ou e
iame e o 3 " a i e iame e o 3" a gage (ou e iame e o "
a i e iame e o 1 " was use
e sc ee s as ic w a a g ass s ecime s i es we e use as e co ec i g
su ace Mos o e sc ee s a as ic w a we e o ai e om e oca g oce y s o e
o a wa e s o e is e a co o os mic osco e s i es (si e 5 75 1 mm om
is e Scie i ic ( i s u g A we e use C A E 4
ME O O OGY
e esea c was s i i o ee is i c oce u es (1 e E ec os i i g ocess (
C a ac e i a io o e o yme as e ec os u i e s usi g a a y ica ec iques a i s ume s a (3 E ec os i i g a c a ac e i a io o e ug a o yme com e
4. El tr p nn n r
(1 e a e e o yme so u io - GA was isso e i E o make -w 1 -w a 15-w so u io s
( So u io ook 3 - 5mi u es o com e e y isso e e o yme (usi g a mag e ic s i i g o a mi i g a a a us a e was e o s a o e ig
(3 e sy i ge wi a gage o -gage iame e ee e o o yme so u io was e a e o e Sy i ge um
( A o -co uc i g as ic u e was co ec e e wee e sy i ge a e ee e
(5 e ee e was ace o i o a y a e e icu a o e e ica su ace (e wi e sc ee as ic i m o g ass s ecime s i e o co ec e e ec os u a o i e s e is a ce e wee e i o e ee e a e e ica su ace was e wee 15- cm
( e e ec o e wi e was co ec e o e sy i ge ee e e e ica su ace g ou e
(7 A ac e sy i ge um o co ui a eake some o e so u io ou oug e sy i ge
( W e e so u io s a e o ow ou o e sy i ge ee e some imes a ea o so u io wou o m a e i ocki g e ow A ai o wee e s was use o c ea e o s uc io
(9 O ce e ow o e so u io was co s a (sy i ge um se a 1 3m1/mi e owe su y was u e o a o age se a 5k
8
(1 A e e ec os i i g a e sam es e owe su y was u e o
(11 e sy i ge um was u e o
(1 e e ec os u sam e was e a e e a e a e o c a ac e i a io a mic osco y
(13 A ma e ia s we e e c ea e
4.2 Ch r t r z t n f th El tr p n b r
i e e ia Sca i g Ca o ime y ( SC — AQ1 is use o e ma a a ysis y measu i g s a i i y oug ea ow (ca ei e e a ea /coo / ea ocess o a ea
ocess
(1 e a e e ec os u i e sam e y cu i g a iece o e i e ma om e g ass s i e
( Usi g SC com u e so wa e ac i a e e coo e u o SC gas
(3 Usi g a a umi um a a e e a a weig i e sam e ace a i o e sam e a a c im e i o o e a
( Usi g e Wi a o io o e co o a se a ame e s o sam e (e em e a u e sca e a e o em e a u e c a ge sam e weig
(5 u sam e g a a sa e g a o i e
( U oa sam e u o SC gas a eac i a e e coo e
e ma G a ime y A a ysis ( GA - AQ5 usi g weig as a measu e o
em e a u e Wi i c easi g em e a u e esi ua mass is os
(1 u o GA gas
( Usi g GA sam e oa i g ock us u ace u o
(3 us sam e u o o o ai access o e a a ce o o ouc a a ce
(4) Once the weigh tray is detached from the balance, tare the weigh tray with empty aluminum pan.
(5) Load sample onto the pan. Using the TGA computer software, attach weigh tray to the balance and run sample.
(6) Graph sample and save on file.
(7) Unload sample, turn off TGA gas.
Scanning Electron Microscopy (SEM) is an electron microscope that uses a beam of focused electrons across an object to produce a high-resolution (three-dimensional) image.
1 Sign the log in book.
2 Using the SEM computer software, log in, record initial parameters, and check with log in book.
3 Prepare sample, using tweezers, gloves, and loading disk.
4 Click vent (Nitrogen gas) to get to atmospheric pressure in sample chamber to load sample.
5 Load sample (still wearing gloves) and press pump. Make sure door to sample chamber is shut. At this point the gloves can be taken off.
6 Using right joint stick, bring stage up to I1 mm (or two finger width) distance between the sample and the power piece.
7 Apply voltage. Using tool bar, use center point locator, magnification, focus, and stigmation to obtain the sample area of investigation.
8 Scan sample area under investigation and save in file.
9 Turn off voltage and write down final parameters in log in book.
10 Turn vent on, and unload sample (wear gloves).
11 Turn pump on and log off software. 40
4.3 Electrospinning and Characterization of Drug and Polymer Complex
Characterization was done with the SEM to determine if the drug had any affect on the polymer diameter or morphology.
DSC was run to see any changes in temperature based on the presence of the drug.
TGA was run to determine if the amount of mass loss in the polymer sample and at what temperature degradation might take place.
All three would determine if the process of putting the drug on the polymer worked. It was the goal of this research to see if the drug was present in the polymer.
Further analysis will have to be done on the amount, release rate, and functionality of the drug in the electrospun polymer. Also whether the electrospinning had any affect on drug performance. CHAPTER 5
RESULTS
C oosi g e so e was a c ucia s e i e ocess I o e ai wi e e ec os i i g ec ique me y e e c o i e (MeC1 was use as e so e o a 75/ 5
GA Ow ac ice sam e is was o e so e o e use o e ug
e i e y sys em (w ic was c ose o e e a y o u a [ ] y a us i g e
ec ique a o ai i g a equa e i e s e ac ua e e ime a sam es we e ma e
W e usi g a ig y o a i e so e ce ai me o s ee e o e i o uce a e g o es eyeg asses mask (co e i g ose a mou a a ume oo
(w e e a e mi i g occu e was use W i e mi i g e o yme a e so e usi g 1 mI o so e a a ious g ams o o yme (1 g ams o Ow g ams o
w I5g ams o 15w a g ams o w ue o e a u e o e so e 5
( y o ume o so e s ow y issi a e om a I m1 so u io O ce e o yme was mi e e sam es so u io s we e sea e o e e u e e a o a io o so e
( e e o e c ea i g a mo e co ce a e so u io a o igi a y i e e A e e i g
e so e se e o e ig e e ec os i i g ocess comme ce
e sam es ma e we e a Ow o / a 5/15 a a w o /
5/15 a 75/ 5 o yme I e i i ia s ages o e e ec os i i g ocess a s i es o
a ious sam e weig so u io s we e a se o i e s (o y o e s we e ese w e s ecime s we e e a ua e wi e o ica mic osco e (OM O e e ec a io o is o se a io is a e so u io was o iscous e oug a ee e a o ge ime o se e
A e a ew ays (3-5 ays e / - Ow emo s a e i e s o e s i e wi mu i e ea s i e s a s (usi g e OM a e 5/15- Ow s owe a ew s a s
41 42
but mostly atomized droplets (using the OM). After a two-week period, the 80/20-
1 Owt% was completely solid, where the THE completely evaporated. The 85/I5-10wt%
solution was had very high viscosity and therefore could not undergo the electrospinning
process. The 85/15-6wt%, 80/20-6wt%, and 75/25-6wt% still had low enough viscosity
to electrospin. At the two-week period, the 85/15-6wt% seemed really viscous (not
quantified, just a physical observation) and there was some doubt as to whether it would
pump through the syringe. Surprisingly, the solution provided a small diameter fiber
network with no beading. Under optical microscopy, there were sections of the fiber that
were larger and flatter while other sections appeared round and thinner. The polymer
samples proved to be sustained at a max time limit of two weeks, in comparison to the
MeCl, which had only a one-week longevity period, but produced fibers within a day of
mixture. This may indicate that the entanglement of the polymers can be sustained for a
certain range of time depending upon the solvent used. The eventual hardening of
solution is due in part to both entanglement properties and evaporation.
In the process of spinning nanofibers, initially a 20gauge needle was used, but a
22gauge proved useful in creating smaller diameter fibers and providing extra pressure at the point of ejection of the fluid jet. Interestingly, the smaller needle diameter provided by the 22gauge allowed for fiber formation to occur in sample solutions that were creating only droplets with the 20gauge. Smaller diameter fibers were also created by increasing the distance between the tip of the fluid jet and the screen from 15mm to
—25mm. Gradually as time increased, with the fibers undergoing entanglement, there was a higher probability for fiber formation, and an increase in density of fiber samples
(observed under the OM [Table 5.I]). 4
The 80/20 1 Owt% samples on 11/25/03, observed under the OM, defined a distinction between beading and density. The 20gauge electrospun sample looked as if there were more beads intertwined in the fiber network than the 22gauge. This was due to the dense network (more fibers per um^2) of fibers by the 20gauge than the 22gauge.
The 22gauge did not produce a thick fiber mat (sheet of fiber sample on slide), therefore beading seemed noticeably less than in the 20gauge slide. In comparison though, based on the umA2 and beading average, the two samples produce the same results.
bl . Fiber Observations Using OM
Date Polymer Wt% Needle size Description 11/19/03 80/20 lOwt% 20gauge Fibers present, but lots of beads 11/19/03 80/20 6wt% 20gauge Droplets 11/19/03 85/15 lOwt% 20gauge Droplets 11/19/03 85/15 6wt% 20gauge Few fibers, mostly droplets 11/19/03 75/25 6wt% 20gauge Droplets 11/20/03 80/20 10wt% 20gauge Lots of fiber formation with beading, formation in 3D, fiber diameter more uniform, beads encapsulated polymer/solvent I 1/20/03 80/20 lOwt% 22gauge Fibers with lots of droplets, fibers not densely packed, organized pattern appearing, uniform diameter 11/20/03 80/20 6wt% 20gauge Droplets 11/20/03 80/20 6wt% 22gauge Some fibers, some droplets 11/20/03 85/15 lOwt% 20gauge Droplets 11/20/03 85/15 lOwt% 22gauge Few fibers, mostly droplets 11/21/03 80/20 I Owt% 20gauge Large amounts of fiber, scattered beading, uniform diameter 11/21/03 80/20 lOwt% 22gauge Fibers with beading 11/21/03 80/20 6wt% 22gauge Fibers with beading 44
bl . i e O se a io s Usi g OM (Co i ue 11/21/03 85/15 lOwt% 20gauge Fiber formation with beading 11/21/03 85/15 1 Owt% 22gauge Fiber formation with beading, not much difference from 20gage 11/21/03 85/15 6wt% 20gauge Few fiber strands, lots of droplets 11/21/03 85/I5 6wt% 22gauge Few fibers, less droplets 11/25/03 80/20 1 Owt% 20gauge Nice fiber network, uniform diameter with beading present 11/25/03 80/20 lOwt% 22gauge Uniform fiber formation, not as dense a sample, beading still exists 11/25/03 80/20 6wt% 20gauge Droplets I1/25/03 80/20 6wt% 22gauge Droplets 11/25/03 85/15 lOwt% 22gauge Uniform small diameter, fiber formation with less beading 11/25/03 85/15 6wt% 22gauge Few fiber formation, some beading, diameter looks smaller than other samples 11/25/03 75/25 6wt% 22gauge Droplets 11/25/03 Glove Multi Multi Using the sample obtained from the gloves worn, sample on the glove showed a branching network of the fibers, larger diameter than all other samples, and densely packed (3D network) 12/01/03 80/20 6wt% 20gauge Droplets 12/01/03 80/20 6wt% 22gauge Droplets 12/01/03 85/15 6wt% 20gauge Large fiber diameter, branching network, no beading 12/01/03 85/15 6wt% 22gauge Nice fiber formation, no beading, sections where fiber diameter gets larger 12/01/03 75/25 6wt% 22gauge Droplets 12/04/03 85/15 1 Owt% 22gauge Extremely large fiber diameter, no beads, areas where diameter thick and gel-like 12/04/03 85/15 6wt% 22gauge Fiber diameter smaller, sections where diameter gets thick and gel-like 12/04/03 75/25 6wt% 22gauge Droplets 45
Incidentally, during the electrospinning process on I2/04/03 the sample 85/I5-
I Owt%, an indirect relationship was noted. After turning off the power source and allowing the syringe to be placed on the collecting screen, solution was still electrospinning out. There was a slight attraction that still existed between the needle tip and the grounded collecting surface which induced fibers to form and be pulled from the solution stream (residual pressure within the syringe still existed even when the syringe pump was turned off. The distance between the syringe needle and the grounded surface was about 0.75mm. Since the fibers formed by this method were large enough to be seen by the naked eye (the size of human hair fibers), the actual circular whipping motion of the fiber was observed. Using the OM, the fibers were large in diameter, but still created a pattern similar to what was observed when the power source was on and the distance between the needle tip and the grounded surface relatively large (in comparison). Also, the glove sample (fibers taken from latex glove collected during electrospinning process) was shown to have interesting morphology, a branching network based on all the fibers collecting on one surface (all different ratios and wt%) and interacting (Figure 5.1). 46
r . a c i g mo o ogy o se e i g o e sam es usi g OM mag i ica io o 5
U o e iew o e e imi a y a a om a e 5 1 e e mi i g w ic o yme
a ios a weig e ce ages o i e e mos u i o m a sma iame e i e s sam es o / - w a 5/15-15w we e use o su seque s u ies as ose s ecime s a a e ossi i i y o o i i g e mos co sis e i e iame e s
I i ia y as o se e e o e e so u io s i o s i ou a o i e s u o y o e s we e ese o e s i e A e a week e / - w esu e i some e ec os u
i e s wi a mi u e o o e s a ea i g e so u io s we e e o e ( o e a e y as ic w a i o e o s ee u e ocess o e a o a io a ossi e
o yme se i g I a ou o e week a e mi u e e mo e co ce a e so u io
(15w a ess e a o a io a e ess co ce a e ( w o so u io s s a e ou wi 1 m1 o u a e o e week e 5/15-15w s i a 1 m1 o so e a e / - w a o y 5 m1 o so e Wi e ec ease i so u io o ume u 47
o e si y e / - w was o ac ua y w /w e 5/I5-I5w s u ou e
es iame e i e s om e wo so u io s e / - w o uce a ew s a s (3-
5 u o e same o ume as e 5/15-I5w e e o e e e a g eme ocess is
o a u c io o i c ease co ce a io y e a o a io u y ue e a g eme is is o e y e e a o a io o a ou 5 o so u io om sam e / - w a e
o uc io o a ew i e s o sam es a e same ag- ime e e o e ime is o a
ac o o com a iso
e es esu s o e e ec os i i g ocess i ega s o w a o yme
a ios o ac i e o g yco i e we e e / -1 w a e 5/I5 w ese wo
a ios wou e use i com i a io wi 5 m1 o C U (i c u i g i ue o es e e ec os i i g ocess o e ug
e o ma osage equi e o C U a mi is e e i a e ous y is e wee
15 - mg/m ase o e k ow ackg ou i o ma io a e e ec os u
a o i e s a e a ig e su ace o a ea o o ume a io a e se o ume o e a sma a ea ( e e o e ig e co ce a io o ac i e age a i o e osage (5 mg/m was use A e e i g e sam es es 1 ou s e so u io s we e e ec os u e e
ay wi o y e / -1 w us ug o uci g i e s e 5/I5- w i o
e o owi g we e a ac o s a we e ke co s a ug (5 mg/m o C U a i ue so e ( sy i ge iame e u- u (- 5k sc ee is a ce om sy i ge (I5mm- 5mm a so u io ag- ime (o e ig
I o e o o ai e e ec os u i e s wi e sma es iame e wi ou e i usio o o e s i e wi e amo g em e o owi g was co si e e (1 e sy i ge gauge si e e o age e co ce a io o w sam es a e is a ce 48 between the tip of the syringe and the vertical plate. By keeping the syringe gage small, the voltage large as possible (without having droplets form again due to too high an electric field), the smallest concentration of wt% samples, and increasing the distance between the tip of the syringe and the vertical plate, the fibers below [Figure 5.2,
Appendix L] were observed using SEM analysis.
- GA / 11/19/ 3 1 wiw; gage
E = 1 k Sig a A = M SE Mag = 3 K W = mm Sig a = I e s
Figure 5.2 (a) 80/20-lOwt% 11/19/03 22gauge.
5
After 11/25/03, there was more extrusion of fibers therefore a greater diameter range was noticeable. Different fiber morphologies were observed and beading was less apparent.
E = .00 S n l A = M SE W = 4 S n l = In n
igu e 5 (h) 80/20-10wt% 11/25/03 22gauge.
S n l A = M SE M = . K S n l = In n
igu e 5 (i) 85/15-6wt% 12/01/03 22gauge.
5
igu e 5 ( a (q a e e ese a io s o e g o e sam e e a e g o e was use as a co ec io su ace o a i e s o i e e w a a ios u i g e e ec os i i g ocess e i e s a e a o ous mo o ogy ue o e e a o a io o so e om e su ace (i c easi g o e si e
0p E = .00 S n l A = M SE M = 2 W = 2 S n l = In n
igu e 5 ( g o e sam e-mu i 11/15/ 3
5
E = .00 S n l A = In n W = S n l = In n
igu e 5 (s) 85/15-15wt% 12/17/03 22gauge.
a
a 1 = 13
1 = 17 1
9 m
E = .00 S n l A = In n M = 2.42 K W = S n l = In n
igu e 5 (t) 80/20-10wt% + BCNU 12/18/03 22gauge.
r .2 (u) 80/20-10wt% + BCNU 12/18/03 22gauge.
a 1 = 93 m
= 7E m
r .2 (v) 80/20-10wt% + BCNU 12/18/03 22gauge. 60
Using SEM analysis, the following fiber diameters were measured [Table 5.2].
The samples were also tagged by date to track the settling of the polymers in solution.
The average diameter was obtained by totaling the diameter measurements and dividing by the number of measurements taken.
bl .2 Nanofiber Diameter
Polymer sample Date Diameter measured (in microns) Average diameter description (in microns) 80/20 lOwt% 11/19/03 8.3, 4.4 6.35 20gauge Min = 4.4 Max = 8.3
80/20 6wt% 11/20/03 1.4, 0.4082, 0.2253, 0.7422, 1.4, 0.684 22gauge 0.1177, 0.2746 Min = 0.1177 Max = 4.4
80/20 lOwt% 11/25/03 4.4, 3.9, 2.7, 3.0, 2.4, 0.6026, 2.23 20gauge 0.9197, 0.3171, 1.8 Min = 0.3171 Max = 4.4 80/20 lOwt% 11/25/03 5.5, 3.4, 3.0, 5.8, 4.0, 7.6, 0.7298, 2.91 22gauge 0.9288, 1.3, 0.9288, 0.6634, 1.1 Min = 0.6634 Max = 5.8
85/15 6wt% 12/01/03 4.0, 3.5, 4.0, 5.1, 6.5, 5.1, 3.3 4.5 22gauge Min = 3.3 Max = 6.5 85/15 6wt% 12/01/03 3.7, 5.2, 4.9, 4.0, 3.4, 3.4, 4.3, 4.27 20gauge 4.3, 6.1, 4.6, 3.1 Min = 3.1 Max = 6.1
85/15 lOwt% 12/04/03 7.7, 4.4, 7.2, 6.6, 4.4, 4.6, 4.1, 5.76 22gauge 3.1, 5.0, 8.9, 5.0, 6.9, 3.6, 4.8, Min = 3.1 10.1 Max = 10.1 6
om a e 5 e / -1 w a e 5/15- w o uce e es i e s wi ou co si e i g ime as a ac o Wi ime a ac o i e ocess ( e amou o
ime i akes o e o yme so u io o se e i o e o o uce sma iame e i e s a sam e o / - w a a 5/15-I5w was ma e e sam es we e e ec os u a o se e u e e OM [ a e 5 3]
bl . OM i e O se a io s
Date Polymer Wt% Needle size Description 12/09/03 80/20 8wt% 22gauge Droplets 12/09/03 85/15 15wt% 22gauge Droplets 12/15/03 80/20 8wt% 22gauge Droplets, encapsulation 12/15/03 85/15 15wt% 22gauge Fibers and beading 12/17/03 80/20 8wt% 22gauge Droplets and multiple fibers 12/17/03 85/15 15wt% 22gauge Good fiber formation, droplets, gel-like fibers 12/18/03 80/20 8wt% 22gauge Fibers with uniform diameter, droplets, gel-like 12/18/03 85/15 15wt% 22gauge Fibers and droplets
Ge - ike i e s ook ike i e s i a u s a e o m ey a e e i i e s a e u ook as i e wa s o e i e cou co a se a ge - ike su s a ce ( ig iscosi y wou ou ou E ca su a io is w e e ea s o e i e seem o s ow a ee- ime sio a image wi i e ea i se y e ca su a i g e u -se e o yme o so u io e
5/I5-15w ga e e mos co c e e i e o ma io i a o -wo e s ee a SEM a a ysis was co uc e o i e a e age i e iame e [ a e 5 ] 62
bl .4 5/15-I5w eco e iame e
Polymer sample Date Diameter measured (in microns) Average diameter (in description microns) 85/15 15wt% 12/17/03 5.955, 3.176, 5.161, 9.925, 7.146, 4.933 22gauge 3.1, 5.2, 2.352, 10.6, 2.823, 2.823, Min = 2.352 3.293, 4.2, 3.3 Max = 10.6
om e a a ysis o a e i e iame e s (a e age mi a ma e i e
o ma io e / - Ow mi u e was com i e wi e ug A 5/I5- w us
C U was ma e u o esu s ( o i e o ma io we e o ai e Usi g SEM a a ysis o e / Ow us C U e o owi g iame e s [ a e 5 5] we e measu e a a a e age iame e o 77I mic o s was ca cu a e
bl . Measu e iame e o / -I w + C U
Polymer sample Date Diameter measured (in microns) Average diameter (in description microns) 80/20 1 Owt% 12/18/03 7.4, 4.666, 3.5, 2.625, 2.333, 1.5, 2.771 22gauge 4.666, 2.3, 1.458, 1.9, 1.750, 2.9, Min = 1.3 2.2, 3.421, 2.658, 3.7, 1.955, 3.1, Max = 7.4 2.932, 1.222, 1.3, 1.466
e mo o ogy o e / -1 w us C U sys em was e y i e es i g e
esig ooke e y simi a o ke a a a i ica e ee- ime sio a s uc u e
[ igu e 5 ( (u ]
Usi g e GA a a ysis a sam es wi a wi ou e ug s owe a mass
oss o e wee - [ igu e 5 3 (a ( ] Sam e / 1 w OE CA A a ai GA kim g o e 1 Si e 5 5 mg TGA O e a o Kim e y Me o am u a e 15- ec- 3 19 Comme 11/ 5/ 3 gage I s ume GA 5 5 ui 1
I 1 em e a u e ("C U i e sa 3 7A A I s ume s
Figure 5.3 a TGA of 10% wt DLPLGA nanofiber with — 3% wt mass loss. Sam e / Ow + ug i e C A a a GA kim g o e 1 Si e 3 mg TGA O e a o Kim e y Me o E am u a e 1 - ec- 3 15 5 Comme 1 /1 / 3 gage ug = C U Ow i E I s ume GA Q5 5 ui 1
9
"I" 3 5 7 do 1 em e a u e CC U i e sa 3 7A A I s ume s
Figure 5.3 b TGA. of 10% wt DLPLGA nanoftber with drug BCNU — 8% wt mass loss. 65
The DSC analysis [Figure 5.4 (a) — (d)] showed that the raw polymer and the electrospun polymer had the same Tg (glass transition temperature). A Tm (melting temperature) was not observed on the DSC graphs since the polymer is amorphous. The only change in Tg observed was subject to the presence of the drug in the polymer
[Figure 5.4 (e) and (f)]. Sam e 1 i e C A a a SC1kim g o e 3 Si e 1 1 mg DSC O e a o Ma is Me o ea /Coo / ea u a e 17- ec- 3 17 3 Comme GA aw o yme I s ume SC 1 7 ui
Sam e / Si e 1 1 mg Me o ea /Coo ea Comme GA aw o yme i s ea i g cyc e C e mi u e
-
-
- 1
- 1
i 1 1 1 1 E o U em e a u e (°C U i e s 7A A I s ume s
Figure 5.4 a DSC of 80/20 DLPLGA polymer , First heat cycle. U ;
2
Sam e / O Si e 1 1 mg Me o ea /Coo / ea Comme GA aw o yme
Seco ea i g cyc e C/mi
0 2
0 4
5 5C g -
15 -1 -5 5 loo E o U em e a u e (°C
Figure 5.4 b DSC of 80/20 DLPLGA polymer , Second heat cycle. Sam e / Ow i e CA A a a SC kim g o e Si e 5 mg SC O e a o Kim e y Me o ea /Coo / ea u a e - ec- 3 13 1 Comme a o i e sam e gage a gage a e ma e ia 11/ 5/ 3 I s ume SC 1 7 ui
Sam e / Ow Si e 5 mg Me o ea /Coo / ea Comme a o i e sam e gage a gage a e ma e ia 11/ 3 i s ea i g cyc e C/mi
1 -1 •
- --I
± 5 9 C g
1 1 - 1 - 1
E o i em e a u e (°C U i e sa 3 7A A I s ume s
Figure 5.4 c DSC of 80/20 DLPLGA nanofiber , First heat cycle. 69
The DSC analysis of the nanofiber sample of 80/20-10wt% DLPLGA, second
heating cycle is seen in Figure 5.4 (d). The second heating cycle could not be isolated
due to computer programming errors. However, a Tg was still measured for comparison.
In the second heating run of the 80/20-10wt% polymer plus drug, there it is not clear as to where the actual Tg is, since due to the first heating, the drug had been degraded. The first heating cycle gives more definitive proof of the presence of the drug, before degradation [Figure 5.4 (e) and (0]. Sam e / 1 w i e C A a a SC kim g o e Si e; 5 mg SC O e a o Kim e y Me o ea /Coo / ea u a e - ec- 3 13 1 Comme a o i e sam e gage a gage a e ma e ia 11/ 5/ 3 I s ume SC 1 7 ui
Sam e / Ow Si e; 5 mg Me o ; ea /Coo / ea Comme a o i e sam e gage a gage a e ma e ia 11/ 51 3 Seco ea i g cyc e C/mi •
•
7 1 °C
• - 1 1 1 1 E o em e a u e ( C U i e sa 3 7A A i s ume s Figure 5.4 d DSC of 80/20 DLPLGA nanofiber , Second heat cycle. Sam e / 1 w + ug i e CA A a a SC‘kim g o e 5 Si e 1 mg SC O e a o kim e y Me o ea /Coo / ea u a e 1 - ec- 3 17 1 Comme GA / Ow ug = C U I s ume SC Q1 7 ui 5
Sam e / Ow + ug Si e 1 mg Me o ea /Coo / ea Comme GA / Ow ug = C U i s ea i g cyc e Cimi
U- Co Co -1 1 9 C 1 7 9 1 9 °C
1 1 1 1 em e a u e ( C U i e sa 3 7A A I s ume s Figure 5.4 e .DSC of 80/20 DLPLGA nanofiber with drug BCNU , First heat cycle. Sam e / Ow + ug i e C A a a SC kim g o e 5 Si e 1 mg SC O e a o kim e iy Me o ea /Coo / ea u a e 1 - ec- 3 17 1 Comme GA / Ow ug = C U I s ume SC 1 7 ui
C
U Sam e / 1 w + ug Si e 1 mg Me o ea /Coo / ea Comme GA / Ow ug = C U Seco ea i g cyc e C/mi - -
t g
1
-1 - - - - E o U em e a u e (°C U i e sa 3 7A A i s ume s Figure 5.4 f DSC of 80/20 DL.PLGA nanofiber with drug BCNU, Second heat cycle. C A E
ISCUSSIO
A s u y o e y e Sc oo o iome ica E gi ee i g Scie ce a ea Sys ems
e e U i e si y A GA was e ec os u i E so e i o e o esig e i ea sca o o issue e gi ee i g [ ] e i e iame e a ge was om 5 -
a ome e s
In r n p ll r r n d t n
igu e 1 I c ease is a ce iame e ec eases om 5um o 333 m
e C emica E gi ee i g e a me a i gi ia ec o ai e simi a esu s w e e
e iame e a ge o se e was om Sum o 333 m [ igu e 1] e " o s" o se e
a e same mo o ogy as e g o e sam e o ai e i is esea c w e e e
o yme i e s seeme o a e a c e o o o e a o e
om e SEM a a ysis a e a e age iame e s o sig i ica c a ge i
iame e was o se e A a e age iame e o 91 mic o s was ca cu a e o e
73 74
80/20-lOwt% alone and 2.771 microns for the 80/20 1 Owt% plus BCNU. The only difference observed was in the range of polymer diameter. The 80/20-10wt% alone had a min at 0.6634 microns and a max at 5.8 microns, where the 80/20-10wt% plus BCNU had a min at 1.3 microns and a max at 7.4 microns. The drug-polymer combination allowed for a wider range of polymer diameter, but not necessarily with smaller diameters (in the future, these numbers need to be repeated for consistency). While making the solution samples of 80/20-10wt%, a week was need to allow for entanglement of the polymer in order to electrospin fibers. The drug-polymer solution of the 80/20- lOwt% did not need as much time to settle (entangle) and spun out fibers within a few days. The presence of the drug may have affected the critical time at which entanglement occurred and needs further investigation.
Using TGA of the plain polymer fibers (no drug) over a period of time in comparison to that with the drug combined into it, no change in solvent dissipation or mass ratio was observed. Comparing the TGA of the 80/20-10wt% and the 80/20 lOwt% plus BCNU [Figure 5.3], no significant mass loss difference (difference of —4%) was observed. In both the SEM analysis and the TGA analysis, it can be said based on the observations that the presence of the drug did not affect diameter size or amount of mass loss. Using DSC analysis, the raw polymer was compared to the plain electrospun fibers and showed no change in the Tg (glass transition temperature). This provided proof that the electrospinning process does not affect the polymer's properties.
Also, in comparing the raw polymer with the 80/20 1 Owt% electrospun polymers in both the TGA and DSC, the electrospinning assay did not affect the polymer characteristics. In the DSC graphs [Figure 5.4 (a)-(d)], the 80/20 1 Owt% and the 80/20 75
aw o yme g a s a e same g a ou -53 °C as was e ec e a e o e y
A e i E Com a i g e SC o e / Ow us C U o e / - Ow a o e e e was a sig i ica c a ge i e g [ igu e 5 (c -( ] e g was e o e a 1 9°C a 1 9 °C as is co cu e wi i i gs ou a e e a me o
a macy a a maceu ica ec o ogy (Ma i S ai is c a ge i SC a owe
o e assum io a e ug was ese i e o yme
I a s u y o e y e e a me o a macy a a maceu ica ec o ogy
(Ma i S ai usi g C U e ca su a e o y (D, - ac i e-cog yco i e mic os e es
( a io - 1 5 ug/ o yme a ios e iame e o e mic os e es i c ease a e e ca su a io e icie cy ec ease e C U co e o e mic os e es ec ease
G o se o e o yme [ 3] As a e e e e e icia c a ac e is ic e e ec os u
ug e i e y sys em e eases ig co ce a io s o e ug i sma osage o ms o
e a ge si e e e o e ec easi g e si e e ec s a /o o ici ies a a e ossi e w e a mi is e i g C U Wi ski co ac o e ug i e e ec os u i e a a e gic eac io occu e ea i g o e y o esis a e a o i e s ca su o e
ug u c io a i y muc o ge a i o e ysica o c emica o ms suc as I
u e a a ysis o e amou o ug i e o yme e eg a a io a e ( e ease a e o e ug a e u c io a i y o e ug ee o e e o e A so e mo ecu a a a ysis o e ug a e o yme a e e ec ic ie ( ui ysics e ec s o e so e ee o e i es iga e CHAPTER 7
CONCLUSION
Based in the data gathered, it is possible to obtain small diameter fibers by electrospinning using THF as a solvent. By combining the raw polymer, solvent, and drug in a simple mixing technique, the drug was able to reside in the polymer. This was evident when performing a DSC analysis and comparing the raw polymer to the electrospun fibers and the electrospun fibers with drug. Both the TGA analysis and DSC analysis, confirmed that the electrospinning process does not affect the polymer Tg or mass lost. The only characteristic that varied was in the morphology. The SEM analysis showed that the presence of the drug did not affect the diameter size of the fiber or the mass loss, but does affect the Tg.
Further analysis should be done to assess the amount of the drug present in the polymer, the functionality of the drug, shelf life of polymer drug delivery system, the release rate of the drug, and how to get the smaller fiber diameter. However diameter is dependent upon the problem, whether dealing with AVMs or tumors. If designing the system for tumors, the diameter should be under 8 microns, due to catheter diameter constraints. If the system's purpose is to plug up AVMs, then the diameter could be much larger, around I0-15 microns. The sample run for the electrospinning process involved the usage of MeCl, which provided fibers the day after mixing, whereas the
THF did not. Testing out different solvents may help in making small diameter fibers within an allotted time constraint. Other solvents to look at would be chloroform and ethyl acetate, both dissolve BCNU and PLGA. A different drug may be used in place of
BCNU (ex. tumor necrosis factor alpha, protein-tyrosine kinase inhibitors) and the effects
76 77 o e o yme c a ac e is ics i es iga e A so e e was a a ie y o ysica mo o ogy a was o se e a c i g o s k o s a o es a ee se i s uc io s o ow o ma i u a e a gua a ee a ce ai s uc u e APPENDIX A
CHARACTERISTICS OF RESPECTIVE RATIOS OF PLA AND PLGA
es ec i e a ios co i u e o mec a ica oug ess a s e g ou i ei e e c ys a i e o e semic ys a i e o m
o yme Me i g G ass a s em Mo u us eg a (°C (°C (G a a ime (mo GA 5- 3 35- 7 -I A 173-17 - 5 7 A Amo ous 55- 1 9 I -1 GA- MC /A /A -1 5/15 Amo ous 5 -55 5- G 75/ 5 Amo ous 5 -55 -5 G 5/35 Amo ous 5-5 3- G 5 /5 Amo ous 5-5 I- G a = e si e o e u a mo u us = ime o com e e mass oss a e a so e e s o geome y [1 ]
78 APPENDIX B
BIOCOMPATIBILITY OF LACTIDE/GLYCOLIDE COPOLYMERS
Considerations on biocompatibility, the reaction of the patient to the material, and the effectiveness of the drug delivery system.
Lactide/Glycolide Copolymers: Review on Toxicity, Biocompatibility and
Clinical Applications (data up to 1999)
1 Three cyclic monomers - glycolide, lactide, and c-caprolactone
2 Biodegradation of the aliphatic polyesters occurs by bulk erosion. The
polylactide/polyglycolide polymer chains are cleaved by hydrolysis to form
monomeric acids and are eliminated from the body through the Krebs cycle, primarily
as carbon dioxide and water in urine. Because the rate of hydrolysis of the polymer
chain is dependent only on significant changes in temperature and pH or presence of
catalyst, a very little difference is observed in rate of degradation at different body
sites (Brandt et al. 1984, Cutright & Hunsuck I97I).
3 The role of enzymatic involvement in the biodegradation of the
polylactide/polyglycolide polymer has been somewhat controversial. Most early
literature conclude that bioerosion of these materials occurred strictly through
hydrolysis with no enzymatic involvement (Puleo et al. 1998). Other investigators
suggest that enzymes do ply a significant role in the breakdown of the breakdown of
the lactgide/glycolide materials (Fukuzaki et al. 1990, Reed I978, Williams I977-
79 80 19 I Muc o is s ecu a io is ase u o e i e e ces o se e e wee i
i o a i i o eg a a io a es i is su ose a a i e e yme i o eme is
e ec e i e ea y s ages wi o yme s i e g assy s a e w e eas e ymes ca
ay a sig i ica o e o o yme s i e u e y s a e ( o a e a 19
4 I c ease y o i ia a a ge su ace a ea a a ig e o o io o mo ome s
omo e io ogica eg a a io ( akamu a e a I9 9 e wa e u ake i o e
o yme is a so i ue ce y e a io o c ys a i e o amo ous egio s i
ge e a amou ous egio s a e mo e easi y a ec e y y o ysis (Asa o e a
19 9 e a io o c ys a i e o amo ous egio s i e o yme is e e e
o o e ysio ogica e i o me a o e s oic iome y o e mo ome s I
com a iso o - ac ic aci - ac ic aci i c eases e e e o e amo ous
egio s (E g e e g & Ko I99I Woo 19 a co o yme s om ac ic a
g yco ic aci s a e mo e easi y eg a a e a ei es ec i e u e o yme s (C aig
e a 1975
[19] APPENDIX C
MECHANICAL PROPERTIES OF MEDISORB® POLYMERS
Summa i e mec a ica o e ies o o yme o ai e om A ke mes
Ar e Mt ariti Bolk MEW Al r f r r r pf rr rr j S n th t l v r immagomiim eiciiwi s
GE E A MEC A 1CA O E IES
o yme s 5 5 1 1 (Cus om 9 1 / (Cus om o mu a io 5 o y - ac i e / 1 o y - ac i e 1 o y 1- ac i e 9 o y- - ac i e / 5 g yco i e 1 - ac i e eak s ess si 9 1 73 3 7 1 s ai a eak 5 5 5 5 5 Yie s ess si 371 7 7 1 s ai a yie 5 1 3 7 9 5 Mo u us si 1 9 3 7 17 1 7 1
MEC A ICA O E IES O O Y- - AC I E / CA O AC O E CO O YME S
o yme - ac i e/ca o ac o e 5 /5 75/ 5 5/15 9 /1 95/5 Weig a io • e si e S e g si
A ma 1 3 5 3 9 A 1 79 1 o e o e A 3 95 15 o e o e E o ga io o Yie 1 1 1 o ai u e 1 5 1 1 Mo u us K si 1 5 3 1 7 1 5 S o e - a ess 5 5 7 91 95 S eci ic g a i y 1 1 1 3 1 5 1 Com essio mo i g 73 — 13 13 +/- 15 1 +/- 1 1 5 +/- 5 1 5 +/- 5 em e a u e °C
MEC A ICA O E IES O O Y- - AC 1 E / CA O AC O E CO O YME S
o yme - ac i e/ca o ac o e / 75/ 5 5/15 9 /1 95/5 Weig a io e si e S e g si
A ma 5 13 1555 53 5 93 A 1 5 1555 o e o e A 3 3 33 1 1 o e o e E o ga io o Yie - - - 5 - o ai u e 5 5 7 Mo u us K si 1 1 5 1 135 S o e - a ess 79 95 S eci ic g a i y - 1 1 1 - Com essio mo i g - 1 - 1 - 1 1 em e a u e °C
Customer Servict Tel 513-489-0294 FM' S 1 1- 9-77
81 lkenneS Enr l AScience that Delivers IMAMO 8 0 84 O Y W 8
SO U I I Y C A
MEDISOR13® Ethyl Methylene Chloroform Acetone Dimethyl Tetrahydro- Hexafluoro- acetate chloride formamide furan (THF) Isopropanol (DMF) (HFIP) 100 L (Poly-L-Lactide) ns s s ns ns ns s 100 DL s s s s s s s 8515 DL s s s s s s s 7525 DL s s s s s s s 6535 DL s s s s s s s 5050 DL ss* s s ss* s ss* s Caprolactone (PCL) s s s s s s s 75:25 L-lactide/ PCL s s s s s s s 80:20 DL-lactide/ PCL s s s s s s s 100 PGA ns ns ns ns ns ns s
ns = not soluble ss * = slightly soluble (degree of solubility is dependent on molecular weight or IV) s = soluble
Medisorb® is not soluble in water, ethanol, methanol, silicon oil , ethyl ether and petroleum ether (heptane, hexane etc,).
Customer Service Tel 513-489-0294 nr 4 44 APPENDIX E
THERMAL ANALYSIS DATA OF MEDISORB® POLYMERS
Summarized thermal properties of polymer obtained from Alkermes.
Al r ME IS=6 = • S n th t l v r MA SO A E tr
ME ISO ® O YME g A ME I G OI A A
o yme o mu a io G ass a si io em °C Me i g oi °C
1 GA 1 o yg yco ic aci 35 - 5 - 3
1 1 o y- - ac i e 5 - 173 - 17
9 1 G/ 9 g yco i e / 1 I- ac i e 35 - 5 1 -
1 1 1- ac i e 5 - 55 Amo ous
515 5 - ac i e / 15 g yco i e 5 - 55 Amo ous
75 5 75 - ac i e / 5 g yco i e - 53 Amo ous
535 5 - ac i e / 35 g yco i e 5 - 5 Amo ous
5 5 5 - ac i e / 5 g yco i e 3 - Amo ous
515 / C 5 - ac i e / 15 o yca o ac o e - 5 Amo ous
515 / C 5 - ac i e / 15 o yca o ac o e - 5 Amo ous
75 5 / C 75 - ac i e / 5 o yca o ac o e 13 - Amo ous
1 C 1 o yca o ac o e (- - (- 5
Amo ous o yme s ocess em e a u e a ge 1 — 1 °C
Customer Service l 513-489-0294 Y 1 _ 9_7
83 APPENDIX F
CHEMICAL PROPERTIES OF BCNU
Summa i e c emica o e ies o ug C U
Poly(1,3-bis-p-carboxyphenoxypropane anhydride)
ABRAHAM 3. DOMB AND ROBERT LANGER
ACRONYMS, TRADE NAMES BIODELCPP, Poly(CPP), Poly(CPP.SA)
Polyarthydrides
snit ilY(709{: E 1 yoyc i oc ye i oyc I „ycoo
SAMOR A Pi:Ix:mom Biodegradable polymer fra criatmlled drug delivery in a form of implant or iiiiectible microspheres G bade! -BCNU-Loadtal wafer fur the treatment of brain tumors).
PIROPEitELS SPICEAL 1:sfrifiEST Anhydride copolymers of 1,3 6is-p- carboxyphenoxypropane (CPI') with aliphatic diacids such as sehocic acid (SA) degrade in a physiological medium to ('PP and SA. Matrices of the copolymers loadeti with ili.wilved or dispersed drugs degrade in vino and in vivo to constantly release. the dings for periods from I t.10 weeks.
P1101 UNITA CONTAITKINS .E.
Molecule weight P(CPP-SA)
10 g maul GPC-poly styrene M 3±:20, M U OX5±3 [5 standards dl g i Viscosity 258C, 0X210,9 3 dieltiorortierhane
114 (characteristic absiirrion ott NaCI pellet 1,750, 1,810 111 frequencies) PSA 1,740, 1,770, 1,810 P(CPP-SA) 1,712, 1,773 .P(CPP)
iai a Film on NaC1 pellet PSA 1,739, I,503 PcCPP-SA) 1,723, 1,765, 1,804 P(CPP) 1,712, 1,764
UV (characwristic nut P(CPP-SA), dichloromethane 265 abswptioo wavelength) CPP monomer, I N Na011 solution 265
I 1 •■• • I a 1 1 •I ; CI 1o e
84
8
i io io ama ook ai iy 119 15991 y O ice i e si y ess oe ig s ese e
1A1 3- is- -ca o y e o y o a i a y i e
O A CON i o s A IA E E E C
So u i i y mg m1 " e -SA (C •SA 701 (
ms S C O moi CW
a u o o 3 O o a e 3 1 --- -- i e i yi i i ia i 3 1 Ke o es 1 1 E y ace a e 1 1 A ka es a a e eS 1 1 gi e s 1 I Wa e
Mai i iouwi k C1ICI 3 C K 3 (3 a ums K a a m g o e a ( 5
;1 e a o e ies CC •A SC 1 C mi 1 7 ;5 IMO (3
K „ 359 339 5 511 K 3 33 1 3 7 3 9
k kg 11 Ai 15 7 13 11 9
C ys a i i y ( C SA ow e - ay 1 17 5 1 (3 i ac io 3 1 I
W 35 1 1
Como i e seque ce (C -SA i- M C C 1 9 7 59 1 9 51 (3 is i u io o a i i y u SA-SA 1 3 o a i i y o SA-C 1 3 7 9 A e age ock e g WA 1 3 5 Ikg ee o a om ess 3 7 9 1
S a i i y i c o o o m so u io ( emase i a M (C -SA ( (a y i e i e c a ge e o y w ia io 1 5 •; e o y e i a io 37 C 13 5 1535 7 3 a e co s a Ac i a io e e gy kca mo 3 7 7 7
E osio a e mg I P(CPP-SA). 14 A iX2 mm isc (5 1 I os a e u e 1 7 9;51 1 i 7 37 C SA 3 1 am 86
3 a yi e a a i iu i ook Co y ig i by Oxford Univetsity Press, i A is s e e
o y(1 1- is- -ca o y e o y o ig e anhydridu)
O U Y I $ CO - I IO 5 A A K IM;
Erosion o mm ay -1 M os a e u e ( 7 37 C C -SA 1 s 5 (C -SA 5 5 11 E 1
Elimination in i o e SA (7)
7 days in rat brain 95 21 days i rat brain 64 100
Drug retea.se in vitro ay 13(CI -SA (7 3 C U i isc 3 ( 5 i o e aci i isc 9
ug e ease i i o ay 3 C U isc im a e i 1 (7 a ai
i com u i i i y Com a i g wi uma wai ( Co i a i e wi a i ai co ea musc e su seu a e
Su ie Gui o a M is U iCa i i c a imo e Ma y a USA
E E/ E CE5
1- u o A A ei a - S ai oc i ica Aci 1 1 11991i 1 335 1 3 3 oi „A a M Mo a Sci 31 OM 1 75 5 3 o E ci a Mac omo coi es 9911 71 $ Mi i A ai 1 K i ge Mac o /mks 1191391i 1171 1 I A a K I A s i a Aca Sci USA 9 1199 55 Co e A„ 17 Kwy as a a ago i 1 Ma io ia 111 9533 a7 1 a k e a i ia e a s I 5 191 1k u A 3 S Ai se o i ge a M S a ia - I eig e i Mg I iom ica1 My e e i e y S S a a y Ca ause ei 199 i9 APPENDIX G
GMP ANALYSIS OF 75/25 DLPLGA POLYMER
i is e o uc s eci ica io o Me iso ® o yme 75/ 5 GA
Al r
C rt f t f An l
Medisorb® 7525 DL High IV Finished Product Specification 1751001 t : 0 80 4 2 t f M n f t r : 06 28 00
t Sp f t n lt
I e e iscosi y — ig 7 ig
esi ua Mo ome o a Ma imum 1
- ac i e e o esu 1 5 3 17 g yco i e e o esu 5
Co o yme Mo e a io - ac i e 7 — mo e 75 mo e g yco i e — 3 mo e 5 mo e
ea y Me a s 1 m o a Ma imum 1 m
G ass a si io em e a u e e o g 9 ° C
Mo ecu a Weig Mw e o esu 113 K M e o esu 9 K o y is e si y e o esu 1 9
e es s a associa e c i e ia s eci ie i is Ce i ica e o A a ysis co o m wi e i is e o uc S eci ica io s es a is e y A ke mes Co o e e a eu ics I
e ma u ac u i g qua i y co o a e i o me a eco s associa e wi e a o e e e e ce o o Me iso ® 75 5 ig I a e ee e iewe a ou o e com e e e Qua i y Co o es i g o e o com ies wi e i is e o uc S eci ica io 1751 1 es a is e y A ke mes Co o e e a eu ics e a c was o uce i com ia ce wi cu e Goo M n f t r n ac ices as ey a y o e o uc io o i is e ma e ia o is i u io is o f ma e ia is e ease o is i u io
Qua i y Assu a ce-- ___ Date: g "q-3° Me issa A. G i i Qua i y Assu a ce Associa e
87 APPENDIX H
GMP ANALYSIS OF 85/15 DLPLGA POLYMER
Finished product specification of Medisorb® polymer 85/15 DLPLGA. Mermes
C rt f t f An l M d rb® 8 h I S 8 00
t : W 0 602
t f M n f t r : 02 28 0
t Sp f t n lt
I e e iscosi y - /g 7 /g
esi ua Mo ome s o a ma imum 1
- ac i e e o - ac i e 1 7
g yco i e e o g yco i e 5
Co o yme - ac i e - 9 mo e 5
Co o yme g yco i e 1 - mo e 15
ea y Me a s 1 m o a ma imum 1 m
G ass a si io e o g 5 C em e a u e
Mo ecu a Weig e o Mw 1 3 k
e o M k
e o o y is e si y 1 5
The manufacturing, quality control and environmental records associated with the above referenced lot of Medisorb® 8515.13L High IV have been reviewed and found to be complete. The Quality Control testing of the lot complies with the Finished Product Specification 1851001 established by Alkermes Controlled Therapeutics II. The batch was produced in compliance with currenr Good Manufacturing Practices they apply to the production of finished material for distribution. This lot of material is released for distribution,
Prepared by Date „L Kathy Miller QualityAssu a ce Associa e
Reviewed by Date Melissa Maple Quality Assu a ce Associa e
o W3 59- Page 1 of 1
88 APPENDIX I
NON-GMP ANALYSIS OF 80/20 DLPLGA POLYMER
Finished product specification of Medisorb® polymer 80/20 DLPLGA.
Al r
ANALYTICAL REPORT
Experimental Medisorb® 8020 DL High IV Batch No. 00-141-87 Date of Manufacture: 05/29/01
Description Result
Inherent Viscosity 0.62 dL/g
Reviewed by : _ _ Date :
89 0 REFERENCES
1 "E ec os i i g " Department of Chemical Engineering, Virginia Tech., I
Y M S i M M o ma M e e G C u e ge "E ec os i i g A w i i g ui e ge e a es su mic o o yme i e s " Applied Physics Letters, o 7 um e e ua y 1
3 M M o ma M S i G u e ge M e e "E ec os i i g a e ec ica y o ce e s II A ica io s " Physics of Fluids, o 13 um e Augus I
C uc ko C C e Y S e C Ma i " ocessi g a mic os uc u a c a ac e i a io o o ous iocom a i e o ei o yme i i ms " Elsevier Polymer, 7397-7 7 I999
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