OliGroFinal2 121115

OliGro: A Novel Nanoparticle Drug Delivery Solution for Multiple Sclerosis

OliGro: A Novel Nanoparticle Drug Delivery Solution for Multiple Sclerosis

Marisa Babb, Pierre Chesnot, Emily Levine, Sam Seymour, and Job Shiach

Dept. of Bioengineering, University of California Berkeley, Berkeley, CA 94720

(Dated: 11 December 2015)

Multiple Sclerosis (MS) is a devastating disease with no cure. Current treatments aim to slow progression

and manage symptoms, necessitating continuous care for the remainder of patients’ lives. The need for long

term care is a major contributor to estimated worldwide healthcare costs of up to $144 billion annually. Here,

we describe OliGro, a novel solution to restore motor skills and, consequently, MS patient autonomy through

extended release of therapeutic antibodies. Anti-LINGO-1, an FDA-approved drug to regenerate the myelin

sheath, will be housed within a PEG-PLA block co-polymer hydrogel. This scaffold will be packaged into

a native membrane-cloaked nanoparticle to limit immune response to the particle and facilitate intravenous

delivery. Receptors for myelin-specific molecules in the central nervous system will be used for targeting our

particles, with the secondary effect of competing with T-cells that act to degrade the myelin sheath.

Keywords: Hydrogel, Nanoparticle, Drug Delivery, Multiple Sclerosis

I. INTRODUCTION

Multiple Sclerosis (MS) is an autoimmune disease in

which the insulating myelin that sheathes neuronal ax-

ons in the central nervous system are damaged. This

disrupts the ability of the neurons to communicate and

consequently causes a wide range of physical and mental

symptoms. There is no cure for this debilitating dis-

ease, but current ”disease modifying” therapies aim to

speed recovery from attacks, slow progression, and man-

age symptoms.

There are four patterns of MS with differing symp-

tom progressions and histological manifestations, but in

all cases, T-cells, a type of lymphocyte that aids in the

body’s defense, play an active role. T-cells gain access to

the brain through disrupted or leaky tight junctions in

the blood brain barrier (BBB), which is normally highly

selective in the healthy central nervous system (CNS).

The T-cells recognize the myelin as foreign and attack

it, triggering an extensive inflammatory process that can

further impede axonal transmission.

The name multiple sclerosis refers to the scars that

form in the white matter of the CNS. In the healthy

brain, oligodendrocytes are responsible for creating the

myelin sheath that insulates the conductive axons. As

MS progresses, however, oligodendrocytes are less effec-

tive in rebuilding the myelin sheath, and these succes-

sively less effective attempts at remyelination cause a

scar-like plaque to build up around the damaged regions.

A. Clinical Need

The majority of the research into MS treatments fo-

cuses on a stem cell-based myelin regeneration approach.

While stem cells represent a very promising field, their

use poses potential problems that include ethical con-

cerns, potential immune reaction and tumor formation,

limited differentiation ability, and utilization unknowns

depending on the type of stem cell being used. In ad-

dition, inclusion of stem cells in any therapy introduces

more stringent regulation guidelines and greatly increases

the complexity of the FDA regulatory process. For this

reason, we have decided to take an alternative approach

to the problem.

Taking a look at the economic impact of this disease

helps put it into context on a global scale. With current

OliGro: A Novel Nanoparticle Drug Delivery Solution for Multiple Sclerosis 2

costs reaching $57,500 per patient per year in the United

States, MS is one of the most expensive diseases to treat,

second only to heart failure.1 The estimated 2.5 million

cases worldwide bring the total cost of this disease to

around $144 billion per year. Lowering this staggering

figure is part of our goal with OliGro. Rather than uti-

lizing expensive stem cell therapies, our solution reduces

the direct cost of treatment by relying on polymer syn-

thesis. Even more significantly, our therapy minimizes

assistive care costs by making patients autonomous once

again.

B. Proposed Solution

Here, we propose a non-stem cell-based approach for

regenerating the myelin sheath as well as protecting the

myelin from additional damage through systemic deliv-

ery. With systemic delivery, the biocompatibility of our

material is a significant design challenge; however, we

plan to ”cloak” our nanoparticle within a native cell

membrane that the body recognizes as ”self” and thus

evades an unwanted immune response. Specifically, we

intend to cloak our particle with the isolated membranes

of naive, patient-specific T-cells since they contain the

major histocompatibility complexes that identify cells as

”self”.2 We will also investigate the potential for ”off-the-

shelf” non-patient-derived T-cells for use in later genera-

tions of the therapy, which would be cheaper but poten-

tially less safe. Before culturing cells for membrane iso-

lation, T-cells will be transfected with a vector for mem-

brane expression of one or more molecules to facilitate

effective targeting of our nanoparticles.

In order for OliGro to be effective, the ability to mi-

grate through the BBB is essential. Our primary ap-

proach relies on the small size of the nanoparticle to fa-

cilitate diffusion through the damaged, leaky tight junc-

tions. If this approach is ineffective, we propose an ad-

ditional method for allowing the T-cells to penetrate the

BBB through signaling molecules expressed on the sur-

FIG. 1. Cascade of T-cell transmigration through theblood brain barrier by expressing activated α4β1 inte-grin. From Engelhardt 3 .

face of the cloaking lipid membrane. Early studies of

T-cell migration show that only activated T-cells are ca-

pable of bypassing the healthy BBB in MS patients.3

Further research has shown that the active component on

these infiltrating T-cells is the α4β1 integrin (also known

by antigen CD49D, or as the alpha 4 subunit of the α4β1

receptor). This mediates T-cell attachment to the signal-

ing endothelial cells at the capture phase of the cascade

leading to transmigration through the BBB (Fig. 1).3 If

necessary, we will include this α4β1 integrin in the T-

cell membrane that cloaks our nanoparticle to promote

perfusion into the CNS via the traditional T-cell route

characterized by the disease.

Another design challenge for our therapy is effective

targeting of myelin. This is complicated by the fact that

myelin is expressed in both the central and peripheral

nervous systems. Myelin proteolipid protein (PLP) is

a protein that makes up more than 50% of the CNS

myelin proteins. It is ubiquitous across myelin within

the CNS and is found in very low amounts in the periph-

eral nervous system (0.05% of PNS myelin proteins).4

We propose incorporating anti-PLP into the nanoparticle

membrane as a means of specifically targeting it to CNS

myelin. Additionally, PLP has been identified as one

of several targets of invasive T-cells, and therefore our


particle may have the additional benefit of competitive

displacement of the T-cells, leading to enhanced preven-

tion of additional myelin degeneration.4 Other potential

CNS myelin targets include myelin basic protein (MBP),

oligodendrocyte-specific protein (OSP), and myelin oligo-

dendrocyte glycoprotein (MOG).4

Once our nanoparticles make it past the BBB (Fig.

2 B,D.) and target the myelin sheath, they will deliver

a therapeutic antibody that can aid in the regeneration

of myelin and improvement of nerve signalling. LINGO-

1 (leucine-rich repeat and immunoglobulin-like domain-

containing Nogo receptor-interacting protein 1) is a pro-

tein expressed almost exclusively in CNS neurons and

oligodendrocytes, and acts as a negative regulator of

oligodendrocyte differentiation and myelination5. With

this in mind, we propose to deliver an anti-LINGO-1 an-

tibody into the MS-ravaged axonal landscape to aid in

the promotion of oligodendrocyte differentiation and re-

myelination.

We will consider the potential to increase the efficacy

of our particles through engineering the PLP receptors

and anti-LINGO-1 antibody to have greater affinity for

their ligand and receptor. Greater affinity for PLP may

allow greater competitive displacement of T-cells along

the myelin sheath as well as better targeting of the parti-

cles, while greater affinity of anti-LINGO-1 may improve

the efficacy of the treatment and reduce the frequency of

infusion.

II. MANUFACTURING METHODS AND

VERIFICATION OF DESIGN

While the rationale behind our approach is valid and

based on well-established science, the novelty of this

treatment demands that we optimize a number of de-

sign features. It is likely that this optimization will be a

balancing act, where desired properties of our nanopar-

ticles may be inversely dependent on a single design fea-

ture of the particle. Specifically, testing of our particle is

FIG. 2. Our proposed solution encompasses A.) intra-venous delivery of our therapeutic, cloaked nanoparticle.B.) Once in the bloodstream, the nanoparticle will reachthe leaky blood brain barrier and diffuse through dueto its small size or by the same mechanism as activatedt-cells. C.) The nanoparticle is encapsulated in a naiveT-cell membrane tagged with myelin-specific targetingPLP. Contained inside is the anti-LINGO-1 antibodywithin a PEG-PLA hydrogel scaffold. D.) This nanopar-ticle will attach to the damaged myelin sites of neuronalaxons, and, while the hydrogel scaffold degrades, the an-tibody will diffuse out and promote myelin regenerationthrough oligodendrocyte proliferation.

designed for optimizing physical features of our nanopar-

ticle, such as properties of the polymer scaffold, concen-

tration of targeting receptors, and particle size and dis-

tribution. These physical features will all be optimized

for the desired in vivo properties, most importantly to

allow for infrequent systemic delivery with limited toxic-

ity and off-target effects, with drug delivery for effective

remyelination as the ultimate goal.

It would be naive to claim that efforts to design our

particle will follow a linear trajectory. As experiments

are conducted and analyzed in light of other results, an

iterative verification and validation process will occur in

which it will be necessary to re-assess the physical prop-

erties and biological effects of our nanoparticle. As this

process occurs it will likely be necessary to make changes

to our manufacturing and testing protocols.


A. Hydrogel

Inside our nanoparticle, we expect to use a hydrogel

copolymer scaffold carrying anti-LINGO-1 antibodies in

suspension. Controlled degradation of the hydrogel scaf-

fold will allow for therapeutic release kinetics of the an-

tibody. The primary components of this hydrogel will

be poly(ethylene glycol) (PEG), which has been shown

to effectively resist protein adsorption and therefore be

relatively bioinert.6 The second functional polymer to be

used will be poly(lactic acid) (PLA). When crosslinked

with PEG, this polymer is known to degrade completely

and without toxic effects in primate brains.7 In addition,

PLA has well-documented and predictable degradation

rates on the order of a few weeks.8 Adjusting the PEG-

PLA copolymer ratio can be used to fine tune the degra-

dation rate of our own hydrogel to match maturation

time scales of oligodendrocytes in human embryos, which

is in the range of 4 weeks to 2 months.9,10

1. Hydrogel Production

This hydrogel will be created by physical gelation, or

using thermoresponsive attractive properties of the hy-

drophobic PLA domains in the polymer chains to cause

them to spontaneously fold and create a soft physical

network (Fig. 3). This technique has been explored ex-

tensively in the literature for PEG/PLGA combinations,

but is much less common for PEG/PLA and, therefore,

will require optimization.11 To start, we will photopoly-

merize triblock copolymers of PLA-mPEG-PLA in solu-

tion. We will then add the antibodies to be delivered

and form nanoparticles around this solution (see section

on ”Nanoparticle Formation Through Microfluidic Jet-

ting”). The result will be a nanoparticle solution that

is ready to be injected into the patient, with no further

polymerization required. Once in the body, the tempera-

ture will naturally increase and cause the polymer to gel

and form a scaffold for our therapy.

FIG. 3. Physical gelation of PLA-mPEG-PLA copoly-mer. The polymer chains are initially dissolved in solu-tion and solidify to form a gel at an Upper Critical Solu-tion Temperature (UCST) that is designed to be belowbody temperature.

If, using this approach, we are unable to target a gel

phase transition temperature below 37◦C or obtain a sat-

isfactory degradation rate, we will move to a chemically

bonded copolymer network. This method will involve

photopolymerization, again within nanoparticle (or after

formation of the particle). In this procedure, we will ini-

tially functionalize the PEG with methacrylate groups

to cause crosslinking and mix concentrated solutions of

both PEG and PLA components in a reaction vessel.12

The antibodies will also be added, along with an initia-

tor. We will again form the nanoparticles around this

mixture then wash the nanoparticles, and move them to

PBS. At this point, we will subject them to UV radiation

to chemically polymerize the gel network from the inside.

For initial experimentation, all the materials required

for production can be obtained from Sigma Aldrich. For

the purpose of animal studies, we can buy generic anti-

LINGO-1 solution from the chemical manufacturer for

around $360/100 mg of concentrated solution. However,

the actual anti-LINGO-1 solution that will be used for


human trials will need to be FDA approved. Currently,

there is a therapeutic anti-LINGO-1 antibody named

BIIB033 undergoing phase II clinical trials. If these trials

prove to be successful, we may be able to license BIIB033

in our own therapy.

2. Hydrogel Characterization

Once again, the important properties to optimized for

our hydrogel are degradation rate and a gelation tem-

perature below 37◦C. To alter the degradation rate, we

will vary the weight percentage of PEG in the polymer

chains. It is important to keep in mind that, while we

want the longest duration between infusions possible for

patients, degradation rate will also have to be optimized

for therapeutic release of antibodies. Altering degrada-

tion rate will also change the gelation temperature, so we

will need to vary the total polymer percentage in solution

if counteracting this effect is necessary.

FIG. 4. Phase diagram for PEG/PLGA triblockcopolymer, with different curves representing changingmonomer ratios. By varying monomer ratios for ourPEG/PLA design, we should be able to get similar dataand tweak our formulation so that gelation happens be-low body temperature. Figure from Lee et al. 11

Since our hydrogel will be polymerized inside pre-

formed nanoparticles, we will not be able to characterize

it directly. Instead, we will have to make sample batches

of gel alone. Before any more advanced characterization

techniques, we will inspect the mixture visually and make

sure it does indeed become viscous and form a gel at body

temperature. This is especially important in the case of

the physical gelation procedure because we do not know

how well it will work with PLA instead of PLGA. Some

iteration of varying copolymer ratios will be required to

ensure that the mixture indeed solidifies at a UCST be-

low body temperature of 37 degrees as shown in Figure

4.11 We will also ensure that the LCST is well above body

temperature so that, for example in the case of a fever,

the polymer does not liquefy. We are not so concerned

about achieving a particular modulus past gelation, so we

will let stiffness be a driven parameter and simply mea-

sure it via oscillatory rheometry for purposes of repeata-

bility between batches. If, during in vivo experiments,

we determine that the gel is too liquid and inhibiting

delivery, we will adjust crosslinking density accordingly.

Similarly, we will perform a swell test experiment to un-

derstand how our hydrogel will behave when hardening

inside the particle.

3. Hydrogel In Vitro Studies

In addition to the basic visual inspection of our hydro-

gel already mentioned, we will need to test degradation

rate of the gel, presence of byproducts, protein adsorp-

tion, and diffusion of the antibody. For the first, a simple

degradation assay will be conducted. Gel samples will

be immersed in PBS solution and their weight tracked

over time as they degrade.13 However, it is important to

note that we expect faster degradation in vivo due to

environmental factors that are not present in vitro and

results here will provide more of a relative comparison

than absolute in vivo degradation timeframes. For ex-

ample, whereas degradation will be exclusively hydrolytic


in vitro, we would expect some enzymatic degradation in

vivo that would speed up the process. In addition, al-

though we do not expect any, we can verify that any

byproducts of the degrading hydrogel are not toxic, as

was done in Bjugstad et al. 7 with St. Kitts green mon-

keys.

In order to understand the biocompatibility of our hy-

drogel, we will also want to analyze surface protein ad-

sorption. To do this, hydrogel samples will be immersed

in blood plasma and CSF in vitro, and ellipsometry will

be conducted to measure the thickness of any result-

ing protein layer adsorbed onto their surface. While

ellipsometry is not easy to do with non-reflective sur-

faces, it can be done with hydrogels by using a metallic

substrate.14 Making use of the hydrogels transparency,

the light from the ellipsometer is able to travel through

the protein layer, the gel sample, reflect off the metallic

substrate, and refract again through both the gel and the

protein layer. In the case of our own hydrogel, we may

encounter some difficulty if the gel is too opaque. Since

transparency is a function of where in the sol-gel transi-

tion the temperature is, we could adjust it to get a clear

gel and therefore better results.

The antibodies we expect to use are in the range of

3-9 nm and the mesh size of our hydrogel will need to

be much larger than this in order to allow for diffusion

over time.15,16 Rather than trying to verify this mesh size

using Scanning Electron Microscopy or Nuclear Magnetic

Resonance Spectroscopy, which can be very expensive

techniques and may not be necessary, we believe it would

be sufficient to conduct an in vitro diffusion study. In

this experiment, we will fluorescently tag the antibodies

and form the hydrogel in their solution. We will then

track the movement of antibodies in hydrogel samples

over time.

B. Native T-Cell Membrane Cloaking

We have chosen to cloak the nanoparticles in a na-

tive cell membrane to help disguise them from immune

response.17 Specifically, we have chosen to commandeer

T-cell membranes because we know T-cells can infiltrate

the BBB in MS patients without prompting a systemic

autoimmune response.

1. T-Cell Culture

In our in vivo animal testing, we will need to determine

whether naive (non-activated) T-cell precursors ought to

be collected from spleen or lymph node samples, or if T-

cells collected from blood will suffice. Though it would ul-

timately be simpler to collect autologous T-cells from pa-

tients blood, the T-cells circulating in blood have already

been activated by their TCR-specific antigen, and this

could potentially present a problem for our use. There

are several methods and kits available for collecting naive

T-cells from murine spleen and blood, for example, from

vendors like Miltenyli Biotec or Abcam.18

T-cells will be isolated from other tissue or blood cells

using magnetic-activated cell sorting (MACS) beads from

Miltenyli Biotec. Briefly, cells are incubated with mag-

netic nanoparticles coated with either anti-CD8 or anti-

CD4 antibodies, which bind to CD8+ or CD4+ T-cells

in solution. The solution is then transferred to a column

placed in a strong magnetic field, and the T-cells attached

to magnetic nanoparticles will adhere to the column while

other cells will flow through. T-cells are then collected

in a separate vessel after removing the column from the

magnetic field.

Through our in vitro and in vivo testing, we will assess

whether there is a difference between using CD8+ (cyto-

toxic, MHC-I-selective) T-cell or CD4+ (helper, MHC-

II-selective) T-cell membranes to cloak our nanoparticle.

Both cell types play a role in the pathology of MS, but

it could be potentially more expensive to isolate two cell


FIG. 5. ProImmune Protocol for Magnetic Cell Separa-tion.

types instead of one. Notably, if we are required to dis-

tinguish between naive and active T-cells, naive T-cells

are characterized by the absence of CD25, CD44, and

CD69, while these cytokines are upregulated in activated

T-cells.19 For the purposes of our experimental design

outline, we will proceed with assuming CD8+ T-cells.

Cells will be cultured as outlined in Lewis et al. 18 af-

ter being isolated. Briefly, The isolated T-cells will be

plated and coated with anti-CD28 and anti-CD3 in PBS

at for 24 hours. After 24 hours, IL-7 and IL-2 will be

added. The cells will be grown for 24 hours, harvested,

and replated for 3 days. After which time, the cells will

be transfected with a vector encoding the genes for the

protein which will be incorporated into the nanoparticle

membrane.

2. Gene Delivery

As outlined previously, we have also chosen to incor-

porate an antibody for myelin α-PLP into the isolated

membrane, in addition to potentially adding α4β1 inte-

grin for BBB penetration if necessary. In order to inte-

grate α-PLP and α4β1 into the naive CD8+ T-cells, a

transfection method will be used. Transfection works by

introducing a gene vector into the cell using a chemical,

electrical, or viral delivery method (lipofectamine, elec-

troporation, and lentivirus, respectively). All methods

will be tested to identify which is most effective for the

CD8+ T-cell populations. Once inside the cell, the trans-

fected plasmid will take advantage of the host cells cen-

tral dogma to express the protein of interest using a vec-

tor (pjP008) that has been designed to incorporate pro-

teins into the membrane as described in Protein Science.

MACS will be used to isolate the successful transfections

using antibodies for α4β1 and α-PLP. Immunoprecipi-

tation along with western blot analysis will be used to

confirm incorporation into the cell membrane.

It is possible that proteins endogenous to the naive

T-cell membrane prove to be reactive within the body.

Clustered Regularly Interspaced Short Palindromic Re-

peats (CRISPR)-Cas9 is a protein, which, along with

a guide RNA (gRNA), is capable of inducing a double

strand break in a targeted genes DNA and silencing that

gene.20 Should there be a reactive protein native to naive

T-cells on the membrane, CRISPR/Cas9 genome editing

techniques will be used to knock-out the reactive protein

using the genome editing kit from Clontech. This tech-

nique will be valuable in investigating the potential for

removing markers of ”self” and preventing recognition of

”nonself” (i.e. major histocompatibility complex) if we

are to use engineered generic T-cells for a later version of

our therapy.


C. Nanoparticle

Once we have shown we can effectively culture T-

cells and incorporate proteins of interest into their mem-

branes, we will need to isolate these membranes so we

can package and cloak our nanoparticles. Repeated rapid

freeze-thaw cycles in a low-detergent disruption buffer are

known to effectively lyse cells and begin the process of

membrane isolation. Pelleting at low speeds can remove

cellular debris while keeping lipids in solution. Following

this with washing with buffered solution (pelleting lipids

through ultracentrifugation and resuspension) and son-

ication will further contribute to the isolation of T-cell

precursor membranes and formation of vesicles. In order

to preserve functionality of surface membrane proteins,

some or all steps may require protease inhibitors. Effec-

tive protease inhibition can be assessed through western

blot if necessary. Membrane isolation and vesicle forma-

tion may be sensitive to specific parameters (i.e. number

of freeze-thaw cycles, buffer solution, sonication length

and intensity); the final procedure will need to be op-

timized for effective formation of nanoparticles. If nec-

essary, affinity purification will be possible through the

targeting of native proteins or membrane expression of a

high affinity tag such as polyhistidine in our T-cell trans-

fection process.

For storage, membrane vesicles will be frozen in

buffered solution (stability over time will need to

be assessed). Alternatively, isolated vesicles may be

lyophilized and stored in this dried form.

1. Nanoparticle Formation Through Microfluidic Jetting

For formation of our nanoparticles, suspension of the

isolated lipids will occur in an organic solvent after again

pelleting the bilayers through ultracentrifugation and re-

moving the disruption buffer. The solvent we use will

need to be optimized for the function of our nanoparti-

cles, as organic solvents can denature proteins that we

want to be functional on the surface of our particles for

effective cloaking and targeting.

Microfluidic jetting is a technique that can be fine-

tuned to produce a uniformly sized vesicle through rela-

tively straightforward means. Formation of a lipid bilayer

is accomplished by placing an organic solution contain-

ing lipids into two connected wells. The addition of small

drops of immiscible solution to each well allows for the

formation of a lipid bilayer between the wells. A narrow

inkjet tip is used to jet a second solution across the bi-

layer, causing vesicles to bud off of the bilayer and form

independent vesicles (Fig.6).21

FIG. 6. Nanovesicle formation from Coyne et al. 21

For our application, the solution jetted across the lipid

bilayer to form the nanoparticles will contain our poly-

mer components in solution. To prevent rupture of the

vesicles, it will be important for the osmolarity of the

buffer into which the nanoparticles are being formed (the

immiscible solution used to form the lipid bilayer be-

tween wells) to be similar to that of our polymer compo-

nents. Nanoparticle size can be controlled during jetting

by varying the distance between the inkjet nozzle and

the voltage applied to the inkjets piezoelectric actuator.

Researchers have found that smaller nanovesicles are gen-

erally more stable with smaller diameters.21 However, if

we are unable to form particles of our desired size, we can

consider introducing non-native lipids to the solubilized

cell membrane solution in order to alter packing param-


eters. By introducing lipids with varying chain length or

head group areas, we can change the critical packing pa-

rameter, CPP, where CPP = v/a0IC (v, hydrocarbon tail

volume; a0, optimal head group area; IC , critical chain

length). While we do not want to change the CPP such

that our particles will form micelles, altering the param-

eter slightly will influence the ability of the lipid bilayers

to form particles of various sizes.

An alternative method to achieving cloaking will in-

volve rehydration/resuspension of a mixture of polymer-

ized, dried hydrogel particles and lyophilized membrane

vesicles in the presence of buffered solution. Rehydration

and resuspension in a concentrated environment may im-

prove efficiency in coating of particles, however, this ap-

proach will likely be less precise and may result in signif-

icant waste compared to in-vesicle polymerization.

Dynamic light scattering will provide insight into par-

ticle size uniformity through measuring the scattering

or reflection of light off of the nanoparticles in solution.

We anticipate a uniform size distribution of the particles

based on previous work.21 If necessary, chromatographic

purification based purely on size exclusion may be effec-

tive for desired nanoparticle isolation, however, if this

method is not effective, an experimentally-determined

ionic gradient should prove sufficient for isolation of de-

sired nanoparticles.

III. PERFORMANCE VALIDATION OF DESIGN

Below, we outline many of the in vitro and in vivo

experiments required to assess the performance of our

nanoparticle design. A common mouse model for multi-

ple sclerosis will be used for in vivo experiments before

moving into clinical trials with patients. The experimen-

tal autoimmune encephalitis (EAE) mouse is representa-

tive of many of the disease characteristics found in multi-

ple sclerosis, and is therefore optimal for this testing. To

induce EAE, mice will be injected with myelin oligoden-

drocyte glycoprotein peptide (MOGaa35−55) followed by

immunization using nonviable Mycobacterium tuberculo-

sis, then inject pertussis toxin into the abdomen. While

several other EAE models and methods to induce en-

cephalitis, this particular model has been shown to pro-

vide leakiness in the brain in order to simulate barrier

bypass.22 A power analysis will be conducted in order to

determine the number of mice necessary for the extensive

and diverse testing described.

A. Toxicity and Off-Target Effects

As a systemically delivered antibody-based therapy,

our nanoparticle has significant potential for off-target

effects and in vivo toxicity. The complexity of these ef-

fects renders them difficult to predict, but can in part be

assessed through a variety of in vitro assays. These assays

will provide indications of upstream results that may lead

to undesirable effects when delivered in vivo. Further-

more, these in vitro assays will help indicate whether our

particle will have the opportunity to encounter and mi-

grate through the BBB, rather than total sequestration

through, for example, a thrombotic response. Once the

results of in vitro assays yield satisfactory results, we will

progress into in vivo studies that will show us the local-

ization, or distribution in the body, of our antibody and

provide a variety of downstream toxicity results. These

in vivo tests will also help identify the clearance mech-

anisms of our particle (i.e. hepatic) and whether the

particle is actually reaching the myelin target.

1. Protein Interaction

In order to understand the behavior of our nanoparticle

in the complex environment of the body it is important

to conduct in vitro assays to elucidate the way in which

our nanoparticle associates with other proteins and cells.

These assays will be conducted in conditions that reflect

both blood medium, due to the planned systemic delivery

of our therapy, and cerebrospinal fluid (CSF) medium, to


assess behavior once the particle has crossed the BBB.

These two environments can be simulated in vitro pri-

marily through proper buffer components and protein

concentration. In healthy patients, protein concentra-

tions in CSF compared to blood are very low (about

100-fold lower). However, in MS patients, increases in

CSF concentrations of IgG and albumin are often seen.

Healthy levels of CSF protein have been established by

the Mayo Clinic (Test ID: SFIN), but MS-related in-

creases of IgG vary generally within <10-fold. For our

assays, concentrations of protein at physiological blood

and CSF levels will be conducted, with also a series of

concentrations of up to 100-fold increase of IgG in CSF-

like buffer.

Specifically, to assess protein-nanoparticle interac-

tions, we will incubate our particles with native proteins

in blood-like and CSF-like buffer for a range of times (be-

ginning with 1 minute and potentially up to 24 hours).

Gel electrophoresis under reducing and non-reducing con-

ditions will provide information about the interaction of

our particles with proteins through variation in band mi-

gration through the gel. Under non-reducing conditions,

nanoparticle-protein complexes should represent a higher

MW, slower-migrating band. The necessary controls will

include protein samples incubated without nanoparticles,

and nanoparticles incubated alone without protein. Par-

ticles will likely be selected that have minimal interaction

with proteins.

2. Thrombosis

As we will be treating patients with a potentially

thrombogenic compound, assessing thrombosis in vitro

will be an important first step. Quantification of platelet

aggregation is commonly done in both basic research

and clinical settings through spectrophotometric mea-

surements of turbidity.23,24 Turbidity measurements are

admittedly low-throughput. If we find a need for a higher

throughput assay, there are plate-based colorimetric as-

say kits and protocols available that measure ADP re-

leased from platelets. In general, these assays rely on

conversion of ADP to ATP, after which ATP-dependent

lucerifase activity affects a colorimetric change. For ease

of use and in the interest of time, we will opt for a

kit based assay. The Abcam kit improves upon the

lucerifase-based assay and provides a fluorescent alterna-

tive: For both turbidity and colorimetric assays, throm-

bin should serve as a positive control and the addition of

buffer (PBS) would serve as a sufficient negative control.

We do not expect our nanoparticles to induce rapid clot-

ting and, as such, conducting these assays over a range

of incubation times should provide a sense of the kinetics

of thrombosis.25

3. Immune Response

Although we are cloaking our nanoparticle in a native

membrane, it is still possible the host immune system

may recognize components of our nanoparticle as for-

eign and initiate an immune response.26 To ensure our

nanoparticles truly evade the hosts immune system, we

want to compare the state of the immune system be-

tween both treated and sham-injected EAE and control

animals. The complement cascade is an early indication

of immune response, and we can survey this potential

activation with an ELISA assay for complement compo-

nents (kits for which are commercially available through

Abcam) in mouse plasma or serum. We can also mea-

sure cytokine blood levels with a similar cytokine array

kit (R&D Systems) to determine the relative levels of se-

lected mouse cytokines. We expect that if our nanopar-

ticle does not evoke an immune response, cytokine lev-

els between sham and treated WT mice, and sham and

treated EAE mice should be comparable, respectively.

If we do detect complement activation or a significant

change in cytokine levels in either or both comparisons,

we can follow up with histology to determine the extent

of inflammation. Briefly, animals would be sacrificed in


compliance with approved IACUC protocols and brains

would be dissected out, fixed with 4% paraformaldehyde,

and sectioned for histology according to the protocol out-

lined for the method of choice. Sections would then be

processed through immunohistochemistry with immune-

cell specific antibodies like Iba1 for microglia and GFAP

for astrocyte activation before imaging to visualize the

cellular landscape.

4. In Vivo Localization

Visualizing the localization of our nanoparticle in vivo

will help us to determine if the particle is reaching the

CNS and how the body sequesters and clears the parti-

cles. In order to model the location of our nanoparticle

noninvasively and continuously in vivo, we intend to use

near-infrared imaging techniques to detect its concentra-

tion and location dispersion. The dosing scheme will be

similar to the above immune response study, and may in-

clude the same animals. We will use a fiber optic device

that measures the signal intensities to determine kinet-

ics of our antibodies conjugated with NIR fluorospheres

or lipophilic tracers (available from Thermo Fisher and

other suppliers).27 This imaging technique will allow us

at least a 48 hour window, evaluated every 4 hours. Given

the fluorescent intensities, we would be able to determine

the localization of the antibodies systemically and at the

target CNS.28 We expect that for the first few hours post

intravenous injection, the drug will travel through the

body systemically before reaching the BBB and entering

the CNS, targeting the myelin PLP. As seen in the Figure

7, we similarly expect our nanoparticle to cluster in the

CNS, and specifically at the damaged axons. At set time

periods, animals will be sacrificed and various samples of

blood and tissue will be obtained for fluorescent quan-

tification. Blood samples may be taken at more frequent

intervals. Tissue samples will include at minimum brain,

liver, kidney and major blood vessels.

FIG. 7. In vivo NIR optical imaging of HER2-positivetumor xenograft mice post treatment with Alexa Fluor750-labeled conjugates. From Lee et al. 28

5. In Vivo Toxicity Monitoring

We plan for OliGro to deliver therapeutic effects over

the course of 1-2 months. As a result, we need to conduct

studies in the long term to determine if the drug contin-

ues to be viable and remains in large enough concentra-

tions in order to extend therapeutic effects. In order to

conduct these long term studies, we will inject labeled an-

tibodies with a different signal intensity than originally

used that targets our specific therapeutic antibody for up

to 2 months to verify drug concentration remains viable

for treatment.

In vivo pharmacokinetic and pharmacodynamic

(PKPD) assays will be conducted to assure that our

nanoparticle, delivered in a specified threshold above

expected concentrations, does not have toxic effects in

model organisms. Early studies will use rodents, while

more complex later-stage trials will be conducted in

larger animals. Generally, blood and urine samples ob-

tained at set intervals can be monitored for changes in

levels of biomarkers, such as liver transaminases AST

and ALT for hepatoxocity (increased levels indicate liver

toxicity), and creatinine for kidney function (generally


a ratio of blood creatinine to urine creatinine, with an

increase in blood indicating decreased golumerular fil-

tration). Timepoints and duration will need to be set

depending on initial PKPD assays. Biomarker data will

be normalized to a baseline sample obtained before ad-

ministration of drug, and data will be compared to ani-

mals dosed with saline (sham), vesicles alone (no hydro-

gel contents), and nanoparticles without the therapeutic

antibody. Histology will be conducted on animals after

sacrifice to identify any toxic effects, especially in the

primary organs such as the liver, kidneys and brain.

B. Drug Delivery and Release Kinetics

A critical aspect of OliGro will be controlled release of

the anti-LINGO-1 antibody. To avoid injection fatigue,

which can lead to low patient compliance, we want our

drug to have a therapeutic effect as long as possible, ide-

ally over the course of at least 1-2 months. First, we

want to ensure proper drug delivery to the myelin sheath

by confirming permeability of OliGro through the BBB.

Then, we want to optimize a robust drug release profile.

1. Blood Brain Barrier Permeability

For in vitro simulation of our device across the BBB,

we intend to use microscopy of primary mouse brain

microvascular endothelial cells (pMBMECs) harvested

from a mouse whose BBB has been previously compro-

mised following the EAE model. The monolayer of pMB-

MECs will be placed in a commercially available two-

chamber assay where live action T-cell extravasation can

be imaged during flow. Fluorescently tagged intracel-

lular adhesion molecules (I-CAM) will allow us to de-

termine whether our α4β1 tagged naive T-cell is capa-

ble of rolling, attaching and bypassing the blood brain

barrier.22

Following this experiment, we can confirm BBB pen-

etration in vivo. This will be achieved using two-

FIG. 8. Experimental setup of two-photon intravital flu-orescence videomicroscopy (IVM) live-imaging EAE andcontrol mice spinal cord window for T cell transmigra-tion to CSF and spinal cord. From Coisne, Lyck, andEngelhardt 22 .

photon Intravital fluorescence videomicroscopy (IVM).

Our empty, naive T-cells tagged with α4β1 integrin will

be fluorescently tagged and washed will a buffer prior

to injection into the carotid artery of EAE and control

mice. The surgical window of the spinal cord will im-

aged in real-time, recording the attachment of T-cells to

the endothelial layer and quantifying T-cell infiltration

into the spinal cord.22 We will also conduct this study

using the tagged nanoparticle without the α4β1, as the

size may allow simple diffusion through the leaky bar-

rier. This will allow us to determine which nanoparticle

version is optimal for entry and targeting and bypassing

the BBB. We primarily expect that the nanoparticle can

enter through this simple diffusion method.

2. Antibody Release Kinetics

An anti-LINGO-1 antibody called BIIB033 was discov-

ered and has begun to be commercialized by the company

Biogen. Randomized phase I trials verified the safety and

tolerability of anti-LINGO-1 in healthy volunteers and

MS patients,29 and a phase II clinical trial is currently

underway. Initial reports suggest that intravenous doses


above 100 mg/kg in humans could lead to serum con-

centrations that are predicted to have pharmacological

activity; however, we will have to follow the phase II and

III clinical trials to determine the actual efficacious con-

centration for clinical use. We can use 100 mg/kg as a

baseline concentration goal for initial studies.

Commercial vendors of Anti-LINGO-1 antibodies, such

as Sigma Aldrich, ThermoFisher Scientific and Millipore,

cite the antibodys size to be about 83 kDa, which means

we would need to deliver at least 7.26x1017 antibodies per

kg to have an effect. Assuming a hydrodynamic radius of

about 9 nm for anti-LINGO-1 (Pepinsky et al. 30 report a

hydrodynamic radius of 15 nm for the antibody-antigen

complex and Mosyak et al. 31 report a hydrodynamic ra-

dius of 6.2 nm for the LINGO-1 protein), the spherical

volume of a single antibody is 3.05x103 nm3. If we esti-

mate the total volume available within each nanoparticle

to be between 6.55x104-5.24x105 nm3 (based on a 50-

100 nm diameter range for each nanoparticle), we could

fix a maximum of 171 antibodies in each nanoparticle.

With this calculation, we would need to deliver 4.24x1015

nanoparticles per kg to have an effect. Of course, this

does not take into the account the amount of volume

taken up by our hydrogel scaffold within the nanopar-

ticle, and we ought to assume that the majority of the

internal nanoparticle volume will indeed be occupied by

hydrogel. In our experimental designs, we will try to fit

as many antibodies into our hydrogel as possible. We will

have to deliver a greater concentration of nanoparticles

than estimated above, which will highly depend on this

final distribution of antibodies within our hydrogel.

Another crucial consideration in assessing drug deliv-

ery will be the actual rate of drug released from the hy-

drogel over time as it degrades. We do not have to take

the T-cell membrane degradation rate into account be-

cause its half life is extremely small and T-cells generally

have rapid turnover. The majority of drug is released

during the second diffusion phase of a triphasic release

process, and we want to design our hydrogel as such so

this phase lasts at least a month in vivo. Besides con-

trolling the degradation rate of the hydrogel, the rate of

drug release depends on other factors such as: the size

and shape of the scaffold (here, we will test various sizes

of spherical gels) and the porosity, or, degree of cross-

linking within our hydrogel.

To assess drug release kinetics from the hydrogel in

vitro, we can start by measuring drug concentration in

a solution surrounding our drug-studded hydrogel over

time. Gels would be placed in 1 ml of PBS in a multi-

well plate and shaken in a room temperature incubator.

PBS would be collected and replaced, and anti-LINGO-

1 concentration would be measured various time point

over the course of minutes, hours, and days in subsequent

rounds of experiments to complete our concentration over

time curve. We could use ELISA or ultraviolet-visible

spectroscopy (UV-Vis, antibodies generally have a UV

absorption peak near 280 nm) to assess anti-LINGO-1

concentration in collected PBS. It is likely we would be

able to run these tests with a standard IgG, which would

be cheaper than using our specific anti-LINGO-1 anti-

body, and still measure similar release kinetics although

the comparison test would have to be run to ensure this.

We will also want to test drug release kinetics in cell

culture once our baseline is established in well-plates to

ensure the rate of drug release is consistent in a more

physiologically relevant environment.

C. Therapeutic Performance

Finally, it will be important to ensure that our

nanoparticle actually achieves the desired therapeutic ef-

fect that we are seeking. We will start with an in vitro

approach in which we assess axonal recovery and re-

myelination in a co-cell culture of axons and oligoden-

drocytes. Moving on to animal tests, we will want to

survey our mice throughout treatment for regained neu-

rological function alongside looking for oligodendrocyte

proliferation and myelin regeneration. A 0-5 functional


scoring scale is widely used to measure neurological func-

tion of the EAE animal model, with a higher score indi-

cating a more severe disease state. Mice are scored daily

for clinical symptoms of EAE, as follows: 0, healthy; 1,

loss of tail tone; 2, ataxia and/or paresis of hindlimbs;

3, paralysis of hindlimbs and/or paresis of forelimbs; 4,

tetraparalysis; 5, moribund or dead.32

FIG. 9. Multi-compartment neuron-glia co-culture mi-crosystem as outlined in Park et al. 33

1. Remyelination Assessment in Cell Culture

To assess remyelination in vitro, we will start using a

physiologically relevant, multi-compartmental microflu-

idic neuron/glia co-culture as done in Park et al. 33 (Fig.

9). This cell culture will serve as our control, and

we will add active T-cells to a separate co-culture to

model MS. In order to test if our drug can effectively

cause remyelination, either complete (PLP/α4β1 and

anti-LINGO-1), empty (PLP/α4β1), negative (neither

PLP/α4β1 nor anti-LINGO1), or naked (without anti-

LINGO-1) nanoparticles will be added at logarithmic

doses to the two cultures. Testing of the nerve function

and myelination will be carried out using immunohisto-

chemistry, transmission electron microscopy, and whole

cell patch clamp to measure the amount of myelination,

myelin morphology, and conductance of the neuron (G),

respectively.34

We hypothesize the following results:

Healthy MS

CompleteSlightincrease inmyelination

Protection and increasedmyelination to physiologicalnumbers, healthy G

Empty No changeInhibition of myelindegradation until the NPdegrades, decreased G

NakedSlightincrease inmyelination

Increased oligodendrocytenumber, same degradationrate, poor morphology,decreased G

Negative No changeTotal degradation of myelin,very poor G

2. Oligodendrocyte Proliferation and Remyelination In

Vivo

Another pivotal performance experiment is to mea-

sure the extent of oligodendrocyte and anticipated sub-

sequent remyelination in vivo. Does our nanoparticle ac-

tually deliver the anti-LINGO-1 drug effectively enough

to stimulate oligodendrocyte differentiation and remyeli-

nation? Do we also see increased myelination in our

control animals, and does this possibly impede with

healthy neuronal function? We could perform immuno-

histochemistry on sections from animals (both control

and EAE, with nanoparticle and sham treatment) in-

jected intraperitoneally once a day for two weeks with

bromodeoxyuridine (BrdU, 100 mg/kg bw), the thymi-

dine analog incorporated into DNA of dividing cells dur-

ing S-phase and used for mitotic labeling. We would

stain fixed brain sections for oligodendrocyte progenitor

cells with an NG2 antibody, young and mature oligoden-

drocytes with an RIP antibody, and a BrdU antibody.35

If our therapy stimulated oligodendrocyte proliferation,


we would expect to see an increase in BrDU+ cells that

also express NG2+ or RIP+ immunoreactivity in treated

animals.

To complement this experiment, we would also want to

measure a reduction in demyelinated regions in treated

animals. Luxol blue and Bielshowsky are commonly used

to visualize myelin and axons, respectively, under light

microscopy. We would follow the protocol outlined in

Zhang et al. 35 to measure the extent of myelinated re-

gions in sham and treated animals compared to baseline.

We could also assess remyelination post mortem via elec-

tron microscopy. Alternatively, if we have access to an

MRI that could image mice, we could assess and quantify

myelinated regions post treatment in vivo, as is done to

measure clinical diagnosis and prognosis in human MS

patients.36

IV. CONCLUSION

As an entirely novel treatment for MS, our proposed

therapy presents significant hurdles that must be over-

come for success. However, current treatments for MS

are clearly inadequate due to their preventative nature, a

lack of efficacy in some patients and their significant side

effects. As a lifelong disease with expensive pharmaceu-

tical therapies and the need for occupational assistance,

MS also represent a significant financial burden in a world

where healthcare is shifting ever more towards a discus-

sion of quality- and value-based decision making. Our

nanoparticles will not only be cheaper than the current

standard of care, but they will also improve quality of

life and reduce costs by allowing patients to return to au-

tonomy. While challenging, the novelty of our proposed

treatment presents a potential groundbreaking change for

the patients themselves as well as a cost-effective option

for the healthcare system.

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