Evaluation of advanced paclitaxel drug delivery implants for controlled release post surgical treatment against glioblastoma multiforme in the brain

CONTENT 1.1 Motivation, Objectives & Organization 5 2.1 Development of Controlled Release Implants 7 for Chemotherapy to the Brain 2.3 Local Implants for Paclitaxel Delivery 9 3.0 Ev

EVALUATION OF ADVANCED PACLITAXEL DRUG DELIVERY IMPLANTS FOR CONTROLLED RELEASE POST-SURGICAL TREATMENT AGAINST GLIOBLASTOMA MULTIFORME IN THE

BRAIN

ONG YUNG SHENG, BENJAMIN MSc, DIC, BEng (Hons)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING

DEPARTMENT OF CHEMICAL & BIOMOLECULAR

ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE

CONTENT

1.1 Motivation, Objectives & Organization 5

2.1 Development of Controlled Release Implants 7

for Chemotherapy to the Brain

2.3 Local Implants for Paclitaxel Delivery 9

3.0 Evaluation of Paclitaxel Foams for local implants 11

3.1.2 In vitro release of Paclitaxel in PBS 12

3.1.4 Cell Growth, Viability and Apoptotic activity Studies 12

3.1.6 In vivo release of Paclitaxel 14 3.1.7 Intracranial Survivability Analysis 14 3.1.8 In vivo intracranial bio-distribution Studies 15

5.0 Evaluation of Spray-Dried 0.8% Paclitaxel Loaded Discs 32

ACKNOWLEDGEMENTS

The author would like to thank his group members in the drug delivery lab in particularly, Ms Laiyeng Lee and Mr Jingwei Xie for providing the formulations and other technical support Thanks go to Mr Sudhir Hulikal Ranganath, Ms Dawn Ng, Ms Meijia Ng and Mr Junjie Huang for laboratory assistance Also thanks to Ms Fan Lu and A/Prof Lee How Sung, from Dept of Pharmacology, NUS, for carrying out the LCMSMS analysis To A/Prof Gavin Dawe, Ms Alice Ee and Ms Han Siew Peng, Dept of Pharmacology, NUS, for technical training and advise in intracranial surgery, to Ms Kho Jia Yen, NUMI histology Lab, NUS, for consultation on tissue staining and preparations, and A/Prof Ong Wei Yi, Dept of Anatomy, NUS, for his valuable inputs and time on experiment design and concept Final thanks go to Prof Nick Sahinidis, UIUC, and A/Prof Wang Chi-Hwa, NUS as thesis advisors

ABSTRACT

In this thesis, evaluation of three different Paclitaxel controlled release biodegradable implants for post-surgical implantation was carried out The Poly-(DL-lactic-co-glycolic acid) (PLGA) based implants were fabricated in the form of Pressure Quenched Foams, Electro-Hydrodynamic Atomized microparticles and Spray-Dried Discs

Two formulations of foams with different functions were evaluated The formulations were (F1) 5% Paclitaxel loaded PLGA 85:15 foams as the slower but prolonged releasing implant and (F2) 10% Paclitaxel PLGA 50:50 foam for faster drug release Experiments carried out were in vitro cell cultures to compare controlled release from foams vs acute Paclitaxel exposure over 24 hours in terms of cell proliferation response and apoptotic activity We were able to show through the biodistribution in brain tissue experiment that Paclitaxel levels were sustained at ~ 3 mm from the site of implantation over a period of 28 days

Electro-hydrodynamic microparticles were showed to agree with in vitro release within an in vivo environment releasing Paclitaxel for up to 28 days after implantation In an tumor response study, the results suggests enhanced tumor suppression by prolonging time taken to reach max tumor volume of 3,000mm3 by 7 days over the commercial Taxol® product

The in vivo release of Sprayed Discs was carried out and the results show some correlation to the published in Wang et al (2003) [18] The results suggest that in an in vivo environment, sustained release can be achieve for up to 42 days with a peak release into systemic circulation observable

at 21 days after implantation

CHAPTER 1:

INTRODUCTION

One of the main challenges of modern pharmacology has been the delivery of the therapeutic agent to the site of action (where the agent is need) and to reach concentration levels high enough to achieve the desired treatment response Often in many cases, duration of exposure at these levels over prolong periods of time are essential to prevent a relapse back into the diseased state and to provide a sustainable environment for patient recovery Moreover, control

of drug levels below toxicity limits are crucial to prevent/reduce side effects to an acceptable level

Many of these requirements and challenges are not unlike those encountered in the field of chemical engineering The encapsulation drugs in a biodegradable matrix from which the drug can diffuse out from is analogues to chemical reactants diffusing into a catalyst pore By changing constituent block concentrations in the polymer matrix, control of the rate of polymer degradation can be achieved thereby changing rate of release of the drug

Our study focuses on developing controlled releases implants in the form of discs for Paclitaxel to

be surgically inserted to remove remaining tumor cells after a debulking surgery (to remove the main tumor bulk) in the brain The discs are inserted into the cavity (where the resected tumor was removed from) and the wound is closed Over time, the discs will release Paclitaxel into the peripheral tissue up to the durations of more than a month It is hoped that by applying this strategy, tumor cells around the cavity would be eliminated and tumor remission avoided

1.1 MOTIVATION, OBJECTIVES & ORGANIZATION

The long-term vision beyond the scope of this project would be to develop accurate computational models that would aid in the analysis and design of controlled release implants Results from here can be extrapolated to consider synergetic treatments e.g changes in transport of the drug under environment of periodic irradiation therapy which is know to induce interfering physiological changes Irradiation is known to increase blood brain barrier (BBB) permeability which affects drug transport from the implant through drug loss from interstial tissue across BBB into cerebral spinal fluid (CSF) circulation besides drug diluting effects due to CSF coming into the interstial space Modeling such dynamics can help medical practitioners and scientist explain causes for or lack of treatment efficacy of strategies undertaken

To begin on this vision, the goal of this MEng project was to carry out preliminary in vivo experiments to evaluate the treatment with novel Paclitaxel release foam developed by Ms Lai Yeng, a fellow research group member, based on a high pressure quench and rapid solidification

of drug-polymer melt

This thesis presents a step-by-step approach in the analysis of the use of foams for controlled release of Paclitaxel as implants within the brain for the post-surgical treatment of glioblastoma multiforme through combining cell culture and in vivo experiments to evaluate the efficacy of this treatment within the body

Key issues for evaluation of the foams carried out in this thesis involve

(i) In vitro drug release in PBS to examine at the degradation rate of the polymer and hence the drug release profile This section was undertaken by Ms Lee Lai Yeng but is presented

in this thesis for completeness

(ii) In vivo release subcutaneously in mice to obtain release profiles within the body This experiment reconfirms the release profile in a physiologically lipophilic environment This

was thought to be significant since degradation rate of PLGA is likely to change according

to proportions of hydrophilic and lipophilic blocks Moreover, this step evaluates the safety

of the implants against bulk release of the drug resulting in systemic toxicity Weights of the animals were regularly to check this point

(iii) Level of toxicity response of tumor cells cultures to sustained release from the foams through cell growth and relative caspase 3 activity levels Design of controls compared the recovery of the (a) cells to acute exposure (over 24 hrs) with commercial taxol and (b) the experimental foams Experimental design was on the basis of two times the Area Under the Curve (AUC) levels An AUC level is the area under the curve of a plot of axis between Concentration of the drug vs the duration of exposure to the drug and is used here to provide consistency in the design of the control groups with commercial Taxol® This study attempts to show the value of sustained release on a cellular level

(iv) Intracranial biodistribution of the drug in the brain over time This study presents the ability

of the foams to maintain therapeutic levels of paclitaxel at distances away from the implant over a period of one month This is important as it illustrates sustain release and penetration distance of the drug from the site of implantation It also serves as raw data for computational model validation

(v) Intracranial Survivability of tumor-laden rats treated with Paclitaxel laded and placebo (blank PLGA polymer without Paclitaxel) PLGA implants Prolong survivability of the experimental groups over the placebo groups indicate enhanced treatment by the foams

Besides the foams implants, evaluation of two other implant formulations was undertaken Namely, 16.8% Paclitaxel loaded EHDA (ElectroHydroDynamic Atomization) microparticles where we analysed the in vivo release profile as well as tumor volume response and 0.8% Paclitaxel loaded Spray-dried compressed discs in an in vivo release study

CHAPTER 2:

BACKGROUND & LITERATURE REVIEW

This section provides a summary of research in the development of controlled release implants to the brain Section 2.1 provides an outline of the development and challenges to effective treatment, Section 2.2 covers the background of Paclitaxel, which is the chemotherapeutic agent

to be delivered and Section 2.3 will give a review of the research to date specifically of controlled release implants for Paclitaxel

2.1 DEVELOPMENT OF CONTROLLED RELEASE IMPLANTS FOR CHEMOTHERAPY TO THE BRAIN

Over the last three decades there has been a rise in brain cancers like glioblastoma multiforme (GBM), oligodendroglioma, anaplastic astrocytoma, medulloblastoma, and mixed glioma has been on the rise Of these, GBM is the most frequent accounting for 16,797 cases out of 38,453 cases per year of malignant brain tumors between 1973 and 2001 in America alone [1]

The conventional clinical treatment for glioma is by surgical debulking of the accessible tumor from the patient’s brain The amount of tumor removed is often limited by proximity to critical regions for brain function and this presents a risk of tumor re-growth from residual tumor The approach for limiting cancer remission is carried out by conventional systemic post-surgical chemotherapy and radiotherapy courses Unfortunately, these have resulted in limited clinical effectiveness due to restricted transport of chemotherapy agent across the BBB (blood brain barrier) and significant PgP (P-glycoprotein) mediated efflux barrier effects [2] To overcome barriers to effective drug transport, biodegradable controlled-release polymers implants could be surgically located at the site of tumor removal during the debulking surgery Commercial implants

like the Gliadel® Wafer delivering BCNU (Carmustine) has enjoyed limited successes in improving patient survival rates Clinical trials with Gliadel® Wafer vs placebo wafers have been shown to prolong survival in people with newly diagnosed high-grade malignant gliomas (in addition to surgery and radiation) from a median survival of 11.6 months to 13.9 months With recurrent glioblastoma multiforme in addition to surgery, median survival increased to 6.4 months from 4.6 months [3, 4] Since only one third of GBM patients are responsive to BCNU [5] with other Gliadel wafer associated complications like cerebal edema [6], several groups have been working on controlled release for other drugs such as doxorubicin [1] and paclitaxel

Figure A: Chemical Structure of Paclitaxel

2.3 Local Implants for Paclitaxel Delivery

Several studies have been carried out using different materials to achieve controlled release of Paclitaxel from surgical implants Von Eckardstein et al used a nitrosoureas liquid crystalline cubic phase encapsulating carboplatin and paclitaxel and reported reduction in tumor sizes in F98 rat brains Brain tissue concentration of Paclitaxel showed little or no drug in the vicinity of 3 mm beyond 7 days Clinical observations of the same formulations have suggested feasible and safe usage if < 15 mg paclitaxel was used [15, 16]

Li et al used implants based on polyphsophoester p(DAPG-EOP) polymer at 10% drug loading into Polilactofate microspheres which were combined with PEG-100 Brain tissue concentrations after 30 days showed drug concentrations above LD90 (Drug concentration need to kill 90% of tumor cells) of a depth between 5 to 7 mm and enhanced survivability [17]

Wang et al reported the in vitro release profiles of discs released from Poly (DL-lactic-co-glycolic) acid 50:50 (MW 45,000- 75,000) fabricated by spray-drying followed by 2 ton compression, a delay of 15 days before drug release was observed [18]

Elkharraz et al fabricated injectable Poly (DL-lactic-co-glycolic) acid 50:50 based microparticles from oil-in-water extraction/evaporation method and glycerol tripalmitate-based implants with 29 and 60% w/w and showed that release of 73 to 87 % of the encapsulate drug within 7 days in the presence of N,N-Diethylnicotinamide (DENA), a hydrotropic agent for paclitaxel, significantly increase the release of paclitaxel increased due to elevated hydrolysis rate of PLGA polymers and the paclitaxel solubility [19, 20] However, how DENA would be used in drug delivery seemed

to be in question since DENA affects the central nervous system expressed in seizures and behavioral changes [21]

Ho et al., was able to show that a constant zero-order in vitro release of 0.92+/-0.03 pg/day Paclitaxel over 5 days was achievable using Chitosan-egg phosphatidylcholine (chitosan-ePC)

films Inhibition of SKOV-3 (human ovarian adenocarcinoma cell line) proliferation was shown with an ED50 of 211 ng/ml from the films A sustained, zero-order release of Paclitaxel was also seen in vivo over a 2 week period in mice implanted with the films [22]

Ruan et al., developed paclitaxel loaded poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) microspheres and found faster release rates than conventional Poly (DL-lactic-co-glycolic) acid (PLGA), besides being able to provide sustained release of 49.6% of the encapsulated drug after one month [23] In a latter work, paclitaxel was encapsulated with Vitamin E TPGS-emulsified Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, of an average size of 240 nm, prepared by a modified solvent extraction/evaporation techniques with vitamin E

as an emulsifier

Figure B: Chemical Structure for Poly (DL-lactic-co-glycolic) acid

CHAPTER 3:

EVALUATION OF PACLITAXEL FOAMS FOR LOCAL IMPLANTS

3.1 Materials and Methods

3.1.1 Paclitaxel Foam Formulations

Two main formulations were used in this work, the first was based on a 5%w/w Paclitaxel loaded foam with Poly-(DL-lactic-co-glycolic acid) 85:15 (F1) and 10%w/w Paclitaxel loaded foam with Poly-(DL-lactic-co-glycolic acid) 50:50 (F2)

Paclitaxel (Bristol-Myers Squibb, New Brunswick, NJ) was incorporated into the polymer matrix (Poly (DL-lactic-co-glycolic acid 85:15 or Poly (DL-lactic-co-glycolic acid 50:50) (Mol Wt.50,000 – 75,000 and Mol Wt.40,000 – 75,000 respectively) by dissolution in Dichloromethane and spray drying the solution to form microparticles using a Buchi Spray Drier with inlet air flow-rate of 700 L/min, inlet temperature at 70oC, aspirator setting of 100% and a pump rate of 30% The microparticles were collected and freeze dried for 72 hours to remove any residual solvents before being subjected to a high pressure of 70 bar for 45-60 minutes under CO2 within a chamber The high pressure depresses the glass transition temperature of the polymer and allowing dissolution of CO2 gas into the polymer melt Upon rapid decompression at 15 bars per minute, the glass transition temperature of the polymers rise forming gas pockets which escapes leaving interconnecting uniform pores in the foam [24]

The foam was set as a 3 mm diameter disc with a 1 mm thickness by the mold holding the polymer melt in the compression chamber Discs were used for the in vitro, in vivo release profiles and intracranial survivability studies while rods of dimensions of 7 mm length x 1 mm diameter were used for intracranial bio-distribution studies Blank placebo foams were fabricated

in the same way without Paclitaxel

The foam discs and the rods packaged into 2 ml eppendorf tubes before sterilization by gamma irradiation to a dose of 15 kGy Total weight of the foam discs and rods were 3 mg and 2 mg respectively

3.1.2 In vitro release of Paclitaxel in PBS

The foams discs and rods were incubated in 5 ml of PBS (pH 7.4) at a temperature of 37 oC Paclitaxel in PBS was measured by extraction into Dichloromethane and dissolution into HPLC Acetonitrile/water (50/50%) mobile phase The PBS replaced with fresh PBS after every sampling

to ensure that the solubility limit of Paclitaxel in PBS is not reached

3.1.3 Cell Culture Maintenances

The cell line used was a rat glioma cell line (ATCC® Number: CCL-107™), C6 cells, established

by Benda et al [25] and reported to be derived from N-methyl-nitrosourea-transformed rat astrocytes The cells were grown in DMEM (Dulbecco’s Modified Eagle Medium, Sigma) supplemented with 10% Bovine Fetal Serum (Gibco, Invitrogen) and 1% Penicillin/Streptomycin (Gibco, Invitrogen) in a humidified incubator After reaching confluence, the cells were prepared

by washing in PBS and detached from the T-flask with Trypsin-EDTA (Gibco, Invitrogen) The cells were re-suspended to obtain a concentration of 3 x 105 cells/2.5 µL before inoculation into a

75 cm3 T flask containing 15 ml of fresh media

3.1.4 Cell Growth, Viability and Apoptotic activity Studies

Investigation of the cellular response to the Foams was carried out by comparison with three control groups (namely Blanks, 5050_Taxol & 8515_Taxol – see following for explanation) All groups (experimental and controls) were inoculated at a density of 1 x 106 C6Glioma cells into

175 cm3 T-Flask into 50 ml of DMEM culture medium on Day 0 and cell density was counted on Days 4 and 8 The cells in all the flask were allowed 2 days to attach and grow in the flask before the respective foams 5% Paclitaxel Loaded PLGA 85:15 (F1) and 10% Paclitaxel Loaded PLGA 50:50 (F2) were administered into the flask

2 control flasks, 5050_Taxol and 8515_Taxol, received 49 ug and 7 ug of Paclitaxel respectively administered in the form of the commercial Taxol®, obtained from Bristol-Myers Squibb, on day 2 (the two Paclitaxel concentrations were calculated to give an equivalent dosage of 2 x AUC over

24 hrs as the two foams implants would released over 6 days of exposure) After 24 hours, the Paclitaxel laden medium was removed and the cells washed with two rounds of PBS washes before replacement of Paclitaxel-free medium These controls were used to simulate acute exposure to paclitaxel to cells over 24 hours window and observing cellular recovery on Day 4 and 8

Growth curves of the groups were as separate flasks carried out in triplicates and counted by conventional Trypan Blue dye exclusion method in a hemocytometer to obtain cell densities and cell viability on days 2, 4 and 8 3 x 106 cells were collected for caspase 3 activity level measurement using a Caspase-3/CPP32 Fluorometric Assay Kit from Biovision

Caspase 3 activities levels were determined by re-suspending cells in 50 µl of chilled cell lysis buffer The cells were then incubate cells on ice for 10 minutes before adding 50 µl of 2x Reaction Buffer (containing 10 mM DTT) to each sample 5 µl of the 1 mM DEVD-AFC substrate were then added before incubating at 37oC for 2 hours before read samples in a fluorometer equipped with a 405-nm excitation filter and 485-nm emission filter The upon cleavage of the substrate by CPP32 or related caspases, free AFC emits a yellow-green fluorescence (λmax = 505 nm) Absorbances were then compared with controls for comparison on caspase activity response

3.1.5 Animal Care

All experiments were carried out with approval from the National University of Singapore’s Institutional Animal Care and Use Committee (IACUC) and housing and care of animals are provided in accordance with the National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines (Guidelines on the Care and Use of Animals for Scientific Purposes) in

facilities licensed by the Agri-Food and Veterinary Authority of Singapore (AVA) A total of 55 Wistar Rats (obtained from Centre for Animal Resources, CARE, Singapore) were used for the work in this present study The rats were housed in cages and given free access to standard laboratory food and water

The tranquilization, induction and maintaining agent for Wistar Rats was administered as 100 mg Ketamine / kg body weight and 10 mg Xylazine/kg body weight for all surgeries For BALB/c mice, the dose was 75 mg Ketamine/kg body weight and 1 mg Medetomidine/kg body weight

3.1.6 In vivo release of Paclitaxel

In vivo release of Paclitaxel for Foams F1 and F2 was carried out by implanting the foams subcutaneously in BALB/c male mice (beginning at an average weight of 30 g) At fixed time points Day 7, 14, 28, 42, 56 and 70, one experimental group is sacrificed (n = 3 mice) and the foams recovered from the cadaver The foam is re-dissolved in DCM and analyzed for residual Paclitaxel by HPLC The animals were anesthetized with ketamine (75 mg/kg) and medetomidine (1 mg/kg), shaved and scrubbed down with 70% alcohol, dilute Hebis scrub (chlorhexidine) followed with a final scrub with Betadine A small 1 cm incision is made on the lateral flank of the animal and the foam inserted The wound was then sutured up and allowed to heal before removing stitches after 1 week Weights of the groups were taken weekly to check on Paclitaxel related toxicity

The objective of this study was to verify release profile in an in vivo environment and to check for safety for use against implant failure

3.1.7 Intracranial Survivability Analysis

5 x 106 C6 Glioma Tumor cells were injected subcutaneously on the lateral flank of the Wistar rat and allowed to grow Once the tumor had reached the required volume, the animal was sacrificed and the tumor resected from its back and cut into 2 mm pieces for intracranial implantation

For tumor implantation, each rat was anaesthetized, shaved and scrub down with 70% alcohol, dilute Hebis scrub (chlorhexidine) followed with a final scrub with Betadine An incision is made to the scarp of the rat and a 3 mm diameter burr hole was made in the skull 5 mm posterior and 3

mm to the right of the bregma for the animals undergoing intracranial surgery The dura was incised sharply, and the underlying cortex was resected with light suction Hemostasis was obtained by light compression using sterile gauze, and the wound was subsequently irrigated Dissected 2 mm pieces of C6 tumor tissue were implanted in the resection cavity, and the wound was closed by suturing On day 8, the animal was re-anesthetized, the wound reopened and the foam was placed on top of the tumor implant The wound was then re-closed by suturing

A total of 30 rats were used for the survivability analysis, a control placebo (Control, n = 10) received a 3 mm diameter blank PLGA discs and two experimental group were used , one for (i) 5%w/w Paclitaxel loaded PLGA 85:15 discs (F1, n = 10) and (ii) 10% w/w Paclitaxel loaded PLGA 50:50 discs (F2, n =10) The total weight of discs was 3 mg

The rats were weighted once every two days and were sacrificed when they showed signs of persistent anorexia or dehydration, body weight loss of 20%, inability to maintain an upright position or to move, moribundity, lethargy or failure to respond to gentle stimuli, or bloodstained

or mucopurulent discharge from nose or eyes

3.1.8 In vivo intracranial bio-distribution Studies

For biodistribution studies, only (i) 5% w/w Paclitaxel loaded PLGA 85:15 rods (F1, n = 5) and (ii) 10% w/w Paclitaxel loaded PLGA 50:50 rods (F2, n =5)) were used over 3 separate time points (

14, 21 and 28 days) The rods were ~ 1 mm diameter x 7 mm in length and weight 2 ± 0.2 mg each Total number of rats for bio-distribution studies was 60 rats

Each Wistar rat was anaesthetized, shaved and scrub down with 70% alcohol, dilute Hebis scrub (chlorhexidine) followed with a final scrub with Betadine An incision is made to the scarp of the

rat and a 3 mm diameter burr hole was made at 1.5 mm posterior of the bregma and 2 mm left from the midline under stereotaxic control To create a path for inserting the rod, an 18 Gauge needle was inserted to a depth of 7 mm from the brain surface and retracted The foam rod was then inserted completely into the incision created The scarp of the rat was then closed by suturing and the animal allowed to recover while the weights of the animals were monitored daily

At the respective time points, the rats were sacrificed and their brain harvested The brains were immediately frozen, kept at - 80 oC before being sectioned coronally in a rat brain matrix with 1.0

mm thickness Each slice was carefully weighted, homogenized and analyzed for Paclitaxel concentration by Liquid Chromatography Mass Spectrometry (MS) method (LCMSMS) in Dept of Pharmacology, NUS

3.2 Discussion & Results

3.2.1 In Vivo Release Profile

The Paclitaxel release rate was evaluated in vivo to determine the actual rate of drug release within the body’s environment This was used to observe the rate of diffusion of Paclitaxel from the pores of the foam in body fluids and to check for major bulk degradation of the polymer matrix

The 2 foam formulations were first evaluated in vitro in PBS (carried out by Ms Lee Lai Yeng, but shown here for completeness)

Figure 1 shows a comparison between the release between two copolymer (PLGA 50:50 & PLGA 85:15) used starting a common 5% Paclitaxel Loading The data highlights the fact that the release rate of PLGA 50:50 is much faster than the PLGA 85:15 This is because of the higher hydrophilic poly-Lactate content in the copolymer which results in faster molecular weight drop in aqueous PBS than the PLGA 85:15 which has a higher hydrophilic polymer content This understanding formed the basis for selecting these polymer proportions for PLGA We wanted to

offer two options: a fast releasing foam to arrest fast growing tumors besides bringing up the surrounding tissue to a therapeutic concentration and a slower releasing foam to maintain the tissue’s Paclitaxel concentration over a prolong period of time PLGA 50:50 Paclitaxel drug loading was doubled to 10% to rise the absolute amount of Paclitaxel release while PLGA 85:15 drug loading was kept at 5% to meet this objective It is hoped that these two options or the combination of both would provide the surgeon with room for decision making based on the specific circumstances in the operating theatre

The in vitro release profile of the 10% Paclitaxel loaded PLGA 50:50 foams in the form of discs and rod (rods will be used in biodistribution latter) are presented in Figure 2 This figure confirms that the release variability between discs and rods are not significantly difference and allows the use of rods for bio-distribution studies as a model (rods are used due to ease of insertion into the brain and improves survivability from the surgery) No characteristic initial drug burst was observed and the data shows a cumulative 8% drug release after 35 days in PBS In terms of total mass of Paclitaxel releases by day 35, 24.1 µg was released out of 305 µg encapsulated for discs and 18.2 µg out of 230 µg encapsulated in rods

Figure 5A & 5B present the in vivo cumulative % release of Paclitaxel in from 3 mm discs implanted in BALB/c Mice (n = 3 mice) for 5% Drug Loaded PLGA 85:15 & 10% Drug Loaded PLGA 50:50 over 3 weeks respectively for procedure as described in Section 3.1.6 This experiment was carried out to evaluate the safety and drug release behavior of the implant in an

in vivo environment It was observed that in the 5% Drug Loaded PLGA 85:15 foams, 22 wt% of the Paclitaxel released after 28 days while for the 10% Drug Loaded PLGA 50:50 foams, 9 wt%

of the Paclitaxel were released By normalizing both foams to an initial drug loading of 10%, we get about 18 wt% drug release for PLGA 85:15 foams and this suggest no significant difference for in vivo release profile between PLGA 50:50 and PLGA 85:15 Considering the safety in using these implants, the weights of animal were consistently rising throughout the experiments and did

not show signs of Paclitaxel related toxicity, indicating no sign of sudden burst release over the 3 weeks window after implantation

It was observed during the in vitro experiment that the PLGA 50:50 foams showed significant swelling in PBS This was not evident in the discs recovered from the mice The measured diameters of the discs in Figure 6 showed a varying diameter about 3 mm and a possible explanation for the lack of the swelling phenomenon maybe the less hydrophilic environment of the body as compared to PBS

b a

Figure 4: SEM image of pores structures in foams (Mean Pore Diameter = 396.7 um; S D = 160.7um) [24]

Figure 5: In vivo release profile foam discs recovered from BALB/c Mice (n = 3 mice) over 3 weeks from 3

mm foam discs Graph A presents the % cumulative release of Paclitaxel from 5% Paclitaxel loaded PLGA 85:15 foams, Graph B presents the % cumulative release of Paclitaxel from 10% Paclitaxel loaded PLGA 50:50 foams

10% Paclitaxel Loaded PLGA 50:50 Foam

5 % Paclitaxel Loaded PLGA 85:15 Foams

Figure 6: Diameter of foams (F1 & F2) implanted in vivo over 6 weeks No discs were recoverable on

day 42 for PLGA 50:50 foams

3.2.2 In Vitro Cell Proliferation & Apoptotic Activity

The cell proliferation response to the 5% Paclitaxel loaded PLGA 85:15 foams (F1) shows a reduction of 14.6 % and 61.8 % on Day 4 and Day 8 respectively over the Blank control (without treatment) The 8515_Taxol controls arrest cell proliferation and showed a marginal recovery after the 24 hr acute exposure (Paclitaxel concentration to give 2 x AUC for 85:15 foam release over 6 days) from day 2 onwards with a cell growth ratio (Day 8 / Day 4) of 1.12 which was smaller than the ratio for F1 groups which proliferated by 1.52 times over 4 days The apoptotic activity analysis data suggests that between day 4 and 8, the activity drops indicating a recovery trend while activity increase for the F1 group which highlights increasing toxicity

Figure 7: Cell Proliferation Response for Blank controls, 5% Paclitaxel loaded PLGA 85:15 foams (F1)

and 85:15_Taxol, the acute 24 hr exposure control group (Paclitaxel concentration based on 2 x AUC

with 6 day release of PLGA 85:15 foams)

Figure 8: Apoptotic Activity Analysis for Blank controls, 5% Paclitaxel loaded PLGA 85:15 foams (F1)

and 85:15_Taxol, the acute 24 hr exposure control group (Paclitaxel concentration based on 2 x AUC

with 6 day release of PLGA 85:15 foams)

Experiments with 10% Paclitaxel Loaded PLGA 50:50 foams showed a reduction of 35.3% and a 78.9% reduction in cell densities over the blank control groups on Days 4 and 8 Cell growth ratio Day 8 and Day 4 for 5050_Taxol control group was 1.47 compared to 1.11 in flask with the foam This indicated stronger proliferation suppression by the foam This was expressed in an increasing apoptotic activity in the flasks with foams while the activity dropped between Day 4 and 8 showing recovery from the acute treatment

This study highlights the benefits of sustained release of the foams over acute Paclitaxel exposure treatment as observed in conventional IV infusion chemotherapy treatment at over two times a similar AUC as the foams over 8 days The cell cultures with both foams showed tumor growth suppression and an increasing tread of Caspase 3 activation over an experimental window of 4 days while, the acute control treatment suggests evidences of cellular recovery Hence, sustenance of a high level of Paclitaxel within the vicinity of the tumor is critical to prevent cellular recovery and to ensure a commitment to the apoptosis pathway

Figure 9: Cell Proliferation Response for Blank controls, 10% Paclitaxel loaded PLGA 50:50

foams (F2) and 50:50_Taxol, the acute 24 hr exposure control group (Paclitaxel concentration

based on 2 x AUC with 6 day release of PLGA 50:50 foams)

Figure 10: Apoptotic Activity Analysis for Blank controls, 10% Paclitaxel loaded PLGA 50:50

foams (F2) and 50:50_Taxol, the acute 24 hr exposure control group (Paclitaxel concentration

based on 2 x AUC with 6 day release of PLGA 50:50 foams)