A RESEARCH PROPOSAL

ON

FORMULATION, OPTIMIZATION AND IN VITRO EVALUATION OF RESPERIDONE LOADED

NANOSTRUCTURE LIPID CARRIER NASAL SUSPENSION TO ENHANCE BRAIN DELIVERY
FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME
(A proposal for PhD degree)

Submitted by:
Chhitij Thapa
Email:kshitizthapa07@gmail.com
Tel: +9779867317864
March, 2019

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A. Introduction
Glioblastoma is a malignant glial tumor cell of astrocytic origin that is predominant in cerebral hemisphere
constituting at least 50 % of malignant gliomas, the tumor cells may involve in any neuroanatomical
structure and is biologically most aggressive forms of malignancy of CNS (Furnari et al., 2007; Stark et
al., 2005; Louis et al., 2007). It is also known to be glioblastoma multiforme in the previous nomenclature
because of its tissues being heterogeneous in nature which further creates the challenges in understanding
the disease itself.
According to the world Health Organization (WHO) classification of tumors of the central nervous system
(CNS) (Louis et al., 2007) Glioblastoma is classified as a Grade IV astrocytoma (DeAngelis et al., 2001).
The prevalence of the glioblastoma is utmost in older adults specially in 75 – 84 years’ age range while
the incidence is less common in younger children. In most of the case the disease may evolve without pre-
existing genetic predisposition and it is indeed difficult to exactly correlate the link between tumor occur-
rence with possible underlying causes or risk factors. (Krex et al., 2007). Hence, due to the poor prognosis
despite of the recent advancement in disease diagnosis the 5 years’ survival rate is considerably lower (i.e.
less than 5%) and the mean survival time is about 12-15 months (Stark et al., 2005; Mineo et al., 2007) or
even within shorter periods as demonstrated in population based studies (Fisusi, F. et al., 2014). Limited
number of patients known as long-term survivors, survive for more than 3 years (Simpson et al., 1993;
Krex et al. 2007; Bokstein et al. 2008).

Overviewing the serious effects of the disease, there is a need of a promising approach for the effective
delivery of the drug to the targeted tumor sites. The presence of blood-brain barrier (BBB), makes the
brain the least accessible organ for the effective delivery of the drug. Because of the complexity of the
BBB itself, it further hinders the permeation of the drug, since the adjacent brain capillary endothelial
cells are interconnected by tight junctions providing adequate fenestrations and pinocytic vesicles result-
ing in the formation of physical barrier and hindering the diffusion of the small hydrophilic drugs and
large molecules like antibodies and antibody-drug conjugates that is unable to readily diffuse across the
lipid bilayer. Moreover, the presence of efflux transporter residing in luminal capillary endothelial mem-
brane further defies the traverses of lipophilic molecules, specially the small-molecule drugs are the po-
tential targets of these efflux pumps located in the BBB. [Oberoi et al., 2016]. Similarly, the diffusion of
polar and/or large molecules into the brain are further hindered by tight junctions present between the cells
of the choroid plexus and arachnoid epithelia. Tight junctions are highly specific paracellular clefts be-
tween cells of the choroid plexus and arachnoid epithelia. [El-habashy, S. E. et al., 2015]. Also the ab-
sorption of several molecules including peptides and neuropeptides [Witt KA. Et al., 2001] are retarded
by the presence of several degradative enzymes present in the BBB, that results in triggering the biotrans-
formation or inactivation of these molecules.

Considering the potential challenges for delivering the drug through BBB several strategies such as in-
tracerebral, intraventricular, intravascular, and intranasal delivery can be utilized for the effective delivery
while intranasal represents one of the most promising route owing to its potential efficacy, safety, and
compliance (Blasi et al., 2007). From the several findings it is well known that the olfactory region of
nasal mucosa provides a direct connection between the nose and the brain. The olfactory nerve pathways
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are a major component of intranasal delivery and the drug can also be delivered directly to the brain from
the nasal mucosa along the trigeminal neural pathway. Moreover, intranasal delivery may involve the
delivery of the drug through various intranasal sub-pathways: systemic pathway where the drug is ab-
sorbed across the nasal cavity into the systemic circulation and then crosses the BBB into the brain; the
olfactory pathway, where the drug passes through olfactory epithelium (paracellularly and extracellularly)
into the olfactory bulb and further into the brain tissue and into the stem cell factor (SCF); and the trigem-
inal pathway, in which the drug is transported via this nerve system (Khan et al., 2010). Through the
intranasal delivery the drug concentration can be increased resulting the simultaneous reduction of dose
and side effects [Misra et al., 2003]. Moreover, in contrary to the oral route there is rapid onset of action,
since the technique circumvents the first pass hepatic metabolism. It is a non-invasive technique for the
drug delivery to the brain the technique avoids the surgical interventions while certain invasive techniques
result in the disruption of the barrier integrity.

The research will be primarily focused on enhancing the drug level into the brain and then comparing the
drug level with other conventional formulation or routes that involves the systemic circulation of the drug.
So the objective of the research is to formulate the novel formulation with an aim to effectively deliver
the drug to the target site by intranasal route. The formulation will be a second generation lipid nanopar-
ticle also known as nanostructure lipid carrier and the model drug resveratrol will be incorporated within
these lipids based nanoparticles. The formulation is chosen, considering the fact that it is effective in
delivering the drug to the brain because of their rapid uptake by brain, biodegradable and bio acceptable
nature of the polymer and moreover the scale up feasibility and absence of the burst effect makes the
formulation an ideal candidate. Nanoparticles also allow the packaging of the drugs resulting the proper
distribution of the drug throughout the brain and facilitates the controlled release of the drugs as well.
Additionally, the incorporation of the peptide/ligand on the surface of the nanoparticle results BBB tar-
geting. Nanoparticles also offer the improvement in nose to brain drug delivery since the encapsulated
drugs are being protected from the biological or chemical degradation and extracellular transport by the
P-gp-efflux proteins resulting the efficient brain targeting of the drug [Mistry et al., 2009].

Despite of the fact that other forms of the nanoparticles as such polymeric nanoparticles are also under
the limelight of the research and their potential benefits on the drug delivery are also under intense inves-
tigation and their findings are also pointed towards positive direction. However, let’s not avoid the poten-
tial demerits while using these polymeric nanoparticles as a potential drug delivery medium. The polymers
being used during the formulation may be of biodegradable or non-biodegradable nature with synthetic or
natural origin. Hence, chances of interaction of polymers with the reticulo-endothelial system may be
observed resulting the rapid clearance from the blood circulation [Brioschi et al., 2009] while other forms
of lipid carrier like solid lipid nanoparticle and NLC have the higher tendency to escape the reticulo-
endothelial system than compared to polymeric nanoparticles [Shah et al., 2016].

More specifically, when the comparisons are made within the lipid based nanoparticles itself owing to the
drug payloads, drug release profile and physical stability (polymorphic transition) of the loaded drugs
several research concluded that NLC exhibited better result than other forms of lipid Nano-formulations
like Nano-emulsions and solid lipid nanoparticles. The higher drugs payload may be due to the fact that

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NLC consists a lipid phase containing both solid (fat) and spatially different liquid (oil) forming a com-
partment that are entrapped within the solid matrix at a room temperature resulting considerable alteration
in the crystal order and formation of less ordered crystals significantly increasing the drug payloads com-
pared to liquid emulsion and solid lipid nanoparticle (Aqil M. et al., 2018). Therefore, considering the
lipophilic nature, bio-acceptability, prolonged release, feasibility to incorporate both hydrophilic and lip-
ophilic drugs and absence of burst effect makes NLC the promising carriers in the area relevant to brain
targeted delivery systems [Muller et al., 2011]. Moreover, lipid based nanoparticles have increased sys-
temic circulation times and increased tumor retention time by enhanced permeation and retention effect
[Oberoi et al., 2016].

Resveratrol (trans-3,4’,5 tryhidrostilbene) is a polyphenolic compound with promising antioxidant and

anti-inflammatory properties abundantly found in peanuts, graphs, red wine and other fruits and vegeta-
bles. Interestingly, special focus on the chemo preventive and antineoplastic effect of the compound RSV
has been under investigation and the evidence of the research has further testified the potential application
of RSV in the treatment of various cancer cells. The compound is found to be effective in all of the major
stages of carcinogenesis (initiation, promotion and progression) [Singh et al., 2004] and anti-proliferative,
pro-apoptotic and anti-migratory effects were observed in various cancer cells. [Strazzabosco et al., 2017]

Various studies have confirmed that RSV plays major role in regulation of the growth of the glioma cells
since it inhibits protein kinase C enzyme inhibiting Wnt signal pathways. The glioma cells proliferation
is correlated with the increased activity of PKC therefore the inhibition of PKC results in suppression in
the proliferation and induction of apoptosis of glioma cells. In addition, the reductions of nuclear β-catenin
and c-Myc protein levels and lowering of Twist1 and Snail, the major activators of the epithelial-mesen-
chymal transition (EMT) program further adds on suppression in proliferation and increment on incidence
of apoptosis of glioma cells [Strazzabosco et al., 2017]. Moreover, the degree of malignancy is correlated
with the rate of angiogenesis in malignant gliomas and since RSV have the inhibitory effects on the angi-
ogenesis process it further supports the antineoplastic property of RSV. Furthermore, resveratrol alters
different transduction pathways important for GBM proliferation like nuclear factor-kB, Rb-E2F, p53,
phosphatidylinositol 3-kinase/ Akt, and mitogen-activated protein kinase pathways (MAPK) [van Ginkel
et al., 2016] resulting the apoptosis in cancer cells [Miloso M. et al. 1999, Chen et al. 2005].

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B. Research direction
Because of the presence of indistinct boundaries and diffusion of the glioblastoma cells throughout the
brain parenchyma, surgical excision and/or whole- or partial-brain irradiation may result paradoxical ef-
fects. It is glioblastoma stem cells (glioblastoma-initiating cells) that has a pivotal role in the origin and
growth of GBM [Strazzabosco et al., 2017] and even with the surgical treatment, the incidence of tumor
recurrence is higher which is thought to occur by glioblastoma stem cells [Louis DN et al., 2007]. The
survival rate of the patient is found to be significantly improved by the use of temozolomide as a chemo-
therapeutic agent along with the inclusion of radiotherapy and maintenance therapy and is thought to be
best model of treatment against newly diagnosed glioblastoma [Minniti, G. et al., 2009]. But the underly-
ing problem of the therapy lies on maintaining the gaping period (23 days) during the dosing of successive
maintenance dose so as to recover the normal cells from the drugs side effects despite of the fact that
invasion and progression of the remaining tumor cells might takes place [Friedman, H.S. et al., 2000].
Also it is to be noted that the long-term exposure of temozolomide at the therapeutic concentration results
the conversion of glioblastoma cells into glioma stem-like cells which in turn there is a massive expansion
of a newly converted glioma initiating cells population having higher proliferative and invasive potential
which makes more difficult to prevent the recurrence of GBM specially during the rest period without
Temozolimide treatment [Jiao, Y. et al. 2015].

RSV because of its nontoxic and devoid of serious adverse effects and considering the underlying prob-
lems of the conventional therapy our efforts will be on enhancing the delivery of the drug on the brain
glioma cells by using naturally occurring RSV as a model drug. Despite of the ability of RSV to cross
blood brain barrier, evidently the oral bioavailability is considerably lower which might be the result of
the poor aqueous solubility and greater level of drug metabolism hindering the potential effect on the
property of RSV as an antineoplastic agent [van Ginkel et al 2016].

Hence, our research will be more focused on formulating and optimizing the RSV loaded nanostructure
lipid carrier (RSV-NS-NLC) that will be delivered via intranasal route to brain glioma cells, to overcome
the issues relevant to drug solubility and metabolism. The comparative study on the fraction of the drug
reaching the targeted site (brain) of the standard formulation (RSV-NS-NLC) with RSV loaded oral NLC
formulation (RSV-NLC) and RSV intranasal suspension (RSV-NS) will be conducted via different ana-
lytical technical technique (HPLC/LC-MS), the findings may be useful on determining the influence of
route and dosage form in drug delivery. Preformulation studies will be conducted on initial phase where
the solubility of the RSV in different solid lipid (fat), liquid lipid (oil) and the surfactant will be determined
and the best of these will be selected on the basis of maximum solubility. Drug and selected excipient
interaction studies will also be conducted by using several quantitative analytical tools (UV/HPLC) or
qualitative analytical tools (IR-spectroscopy/Differential scanning calorimetry). Formulation of RSV-NS-
NLC and RSV-NLC will be carried on by deploying high pressure homogenization technique [Gupta et
al. 2017]. The optimization will be carried out by using design expert software and study on the effects of
independent variables like solid/liquid lipid ratio, surfactant concentration and homogenization cycle on
the dependent variables like particle size, polydispersity index and entrapment efficiency will be con-
ducted.

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The particle size, polydispersity index and the zeta potential values of the formulations will be analyzed
by the use of dynamic light scattering technique [Aqil M. et al., 2018]. Similarly, the drug entrapment
efficiency will be examined by using suitable analytical tools (UV/HPLC analysis). Various studies on
particle shape and the surface morphology of formulation will be carried by transmission electron micros-
copy and scanning electron microscopy study. The study of the polymorphic effect will be conducted by
x-ray diffraction study or DSC studies.

In addition, several studies will be conducted on accessing and comparing the extent of the drug delivery
to the targeted site (brain). Initially the tumor will be induced on our proposed animal model (Sprague-
dawley rats, 200-250 gm) by xenograft induced model [Abdi, Z. et al 2017]. RSV-NS-NLC and RSV-NS
will be given through intranasal route while RSV-NLC will be delivered to the animal through oral route.
The final effects of the formulation on the animal tumor cells will be observed. The decrease in the tumor
volume will be marked as an indicator in excessing the effectiveness of the drug delivery and the compar-
ative study of the different formulations will be carried on. The average tumor volume will be calculated
as: Tumor volume (cm3) = 0.5ab2, where “a” and “b” will be the longest and shortest cell diameter
[Devineni, D. et al. 1996].

To study the level of drug localization in a brain gamma scintigraphy study will be conducted where each
formulation will be suitably labeled with a compound with a gamma radiation emitting property (techne-
tium-99). The imaging will be performed using single-photon emission computerized tomography
(SPECT) gamma camera where RSV-NS-NLC, RSV-NS and RSV-NLC will be recorded using gamma
camera system and the images will be analyzed with inbuilt software [Chandra et al. 2016].

The study on the amount of the drug reaching the brain i.e. total brain uptake will be further confirmed by
using fluorescent probe (rhodamine B dye) as an indicator. Separate formulations of rhodamine-NS, rho-
damine-NS-NLC and rhodamine-NLC formulations will be prepared and the comparative study will be
conducted by fluorescence imaging [Musumeci T. et al. 2014].

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 C. Candidature plan

Research timeline

Research 2019 2020 2021 2022

timeline Sept- Dec- March June- Sept- Dec- March June- Sept- Dec- March June- Sept- Dec- March June-
Nov Feb -May Aug Nov Feb -May Aug Nov Feb -May Aug Nov Feb -May Aug
Literature re-
view
Preformula-
tion evalua-
tion
Research pro-
posal submis-
sion
PhD proposal
presentation
Annual report
submission
Formulation
and post for-
mulation eval-
uation of
RSV-NS
Optmization,
formulation
and post for-
mulation eval-
uation of
RSV-NLC
Annual report
submission
Optimization,
formulation
and post for-
mulation eval-
uation of
RSV-NS-
NLC
Annual report
submission
Thesis writing

Method and skills

All the chemicals used for the projects will be of analytical grade. Double distilled or deionized water will be
used during the development of the formulations of other chemical reagents. The air and the moisture sensitive
reagents will be suitably handled using flame dried glass ware under the inert gas (nitrogen) atmosphere.
Vortex shaker will be used for the determination of solubility of drug in different oils. High pressure homog-
enizer (BEE international) will be used for the formulation of different dosage forms. Malvern zetasizer will
be used for the determination of particle size, PDI and zeta potential value. IR-spectrophotometer (lumex
instruments) and differential scanning calorimeter (perkinElmer) will be used to perform drug-excipient in-
teraction study. X-ray diffractometer (Malvern panalytical) will be used to detect the drug polymorphic effect.
Transmission electron microscope and scanning electron microscope will be used to determine the shape and
the surface morphology of the formulations. SPECT gamma camera will be used for the analysis of drug

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localization and confocal laser scanning microscope will be used for the determination of the tissue uptake
studies. All the analytical techniques will be performed by the use of UV, HPLC or LC-MS techniques de-
pending upon the feasibility of the study.

D. Field work
For the successful conduction of this research no any field work will be required.

E. Confidentiality and intellectual property

Relevant issues associated on IP are not connected with this project.

F. Approvals
It is free to conduct the research as per the proposed plan of the researcher. No any further special approv-
als are required from any concerned body regarding the continuation of this project.

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G. Bibliography
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