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SUPPLEMENTARY MATERIAL

A. Validation of Extraction and HPLC Method for Measuring siRNA in BrainTissue

We developed a novel extraction protocol allowing full recovery of siRNA to perform

measurements of its distribution across the brain delivered via the inranasal instillation

of the nanoparticles carried Cy-3 labeled siRNA, The major part of this protocol was to

develop a special Extraction Buffer suitable for HPLC analysis with anion-exchange

DNA Pac PA100 column. This column cannot be used with anionic detergents as they

bind irreversible to the column. The known extraction method described in

US2011020100 Patent traditionally uses anionic detergent SDS for extraction of siRNA

but requires its removal before injection to the column by using high concentration of

KCl. Testing of this method in our lab showed that KCL co-precipitates siRNA together

with SDS (Fig S1). The loss of siRNA reaches 95% making such approach unfeasible

for quantitative determination of siRNA delivery to the brain.

Fig S1. Effect of precipitation of hsiRNA by KCl according to standard protocol (patent US2011020100).

1. hsiRNA dissolved in 1 mL ofExtraction Buffer buffer at concentration 1.5 uM;

2. 200 uL of 3M KCl added to hsiRNA solution;

3. Supernatant is separated from pellet after centrifugationat 10 000 g for 2 min.

New Extraction Buffer contains the following components:

25 mM Tris-HCl, pH 7.420mM NaCl1mM EDTA

Elue

nt B

(%

)


0.5% EMPIGEN

Utilization of zwitterionic detergent EMPIGEN in recipe allowed to recover not less than96% of siRNA from the brain tissue.

The protocol of extraction has included the following procedures.

a. Homogenization of brain tissue with Tissue Raptor (Quiagen) in preparedExtraction Buffer with proportion of 10 to 1 (v/wt); for example, 10 uL/mg

b. Centrifugation samples @10,000 rpm x 15 minc. Transfer supernatant to new tubesd. Adding 2 mg/mL of Proteinase K (EpiCentre, Cat# MPRK092) to lyse

proteinse. Lysis of proteins @ 50 C for 20 minf. Centrifugation samples @10,000 rpm x 15 ming. Transfer supernatant to Costar Spin-X Centrifuge tube filters, 0.45 µm

(Cole-Parmer, Cat # UX-01937-40)h. Centrifuge @ 14,000 rpm x 10 mini. Transfer filtered samples into HPLC vials

Analysis was performed on Perking Elmer series 200 HPLC with auto sampler and both UV and Fluorescent detector. Binary gradient pump delivers two eluents A and B programmed to crate gradient suitable for elution of siRNA.Eluent A: 50 mM Tris Buffer, 2 mM EDTA and 50% acetonitrile (pH 8.0)Eluent B: 50 mM Tris Buffer, 2 mM EDTA; 2.6 M NaClO4 and 50% acetonitrile (pH 8.0) The pump was programmed for creation of gradient as depicted on Fig. S2.

Pump B programm

120

100

80

60

40

20

0 0 2 4 6 8 10 12 14 16 18

Time (min)


Fig. S2 Programmed gradient of eluent B.

The typical chromatogram representing siRNA pick with retention time 6.7 min at flow 1 ml/min is depicted by Fig. 3.

Fig. S3 HPLC chromatogram of 6.1 pmol of siRNA

Calibration curve obtained with different concentrations of siRNA depicted by Fig. S4.

PEA

K A

RE

A X

100

0


120

100

y = 15.761x + 1.4282R² = 0.9952

80

60

40

20

00 1 2 3 4 5 6 7

AMOUNT OF SIRNA INJECTED, PMOL

Fig.S4 Calibration curve for quantitative HPLC determination of siRNA extracted from mouse brain.

Validation of HPLC method for quantitative determination of Cy-3siRNA extracted from mouse brain revealed the following parameters (Table S1):

Table S1

Recovery Mean 101.09%

Recovery SD 6.71%

Limit of Detection 1.09 pmol

Limit of Quantitation 3.31 pmol

Dynamic Range 0.9 – 6.1 pmol


B Detailed Method for Synthesis of hsiRNA

Hydrophobically modified siRNA (hsiRNA) against htt (HTT10150) hsiRNA was based

on a previously identified HTT functional targeting site(13).The compounds were

asymmetric, composed of a 15-nucleotide long duplex region with a single-stranded 3′

extension on the guide strand. All bases were modified using alternating 2′-O-methyl /2′-

fluoro modification pattern with additional 14 phosphorothioates incorporated as shown.

The 3′ end of the passenger strand was conjugated to a hydrophobic teg-Chol

(tetraethylene glycol cholesterol). Combination of described modification enables quick,

efficient internalization by primary neurons without requirement for formulation or

electroporation.

Standard solid-phase oligonucleotide synthesi s .Oligonucleotides were synthesized on

an OligoPilot100 Synthesizer following standard protocols. Each synthesis was done at

a 50-200-umole scale using Chole-teg or Unylinker terminus (ChemGenes, Wilmington,

MA) support. The sequence of the hsiRNAHTT used in this study is shown in the Table

S-2 below.

Table S-2

siRNA ID hsiRNA

Accessi onnumber Strand Sequence

fU#mG#fA.mC.fA.mA.fAm.U.fA.mC.fG.mA.fU#mU#fA-NTC N/A Sense

Antise nse

TegCholPmU#fA#mA.fU.mC.fG.mU.fA.mU.fU.mU.fG.mU#fC#mA#fA#mU#fC#mA#fU

hsiRNAHTT

NM_002111 Sense

Antise nse

fC#mA#fG.mU.fA.mA.fA.mG.fA.mG.fA.mU.fU#mA#fA- TegCholPmU#fU#mA.fA.mU.fC.mU.fC.mU.fU.mU.fA.mC#fU#mG#fA#mU#fA#mU#fA

Chemical modifications are designated as follows. “.” – phosphodiester bond, “#” – phosphorothioate bond, “m” – 2’-OMethyl, “f” – 2’-Fluoro, “P” – 5’ Phosphate, “teg- cholesterol” – tetraethylene glycol (teg)-Cholesterol.


Phosphoramidites were prepared as 0.15 M solutions for 2´-O-methyl (ChemGenes,

Wilmington,MA), Cy3 (Gene Pharma, Shanghai, China) and 2´-fluoro (BioAutomation,

Irving, Texas) in ACN.5-(Benzylthio)-1H-tetrazole (BTT) 0.25 M in ACN was used as

coupling activator. Detritylationswere performed using 3% dichloroacetic acid (DCA) in

DCM and capping was done with a 16% N-methylimidazole in THF (CAP A) and

THF:acetic anhydride:2,6-lutidine, (80:10:10, v/v/v) (CAP B) for 15 s. Sulfurizations were

carried out with 0.1 M solution of DDTT in ACN for 3 minutes. Oxidation was performed

using 0.02 M iodine in THF:pyridine:water (70:20:10, v/v/v). Deprotection and

purification of oligonucleotides. Both sense and antisense strands were cleaved and

deprotected using 40% aq.methylamine at 65 °C for 15 minutes. The oligonucleotide

solutions were then cooled in a freezer and dried under vacuum in a Speedvac. The

resulting pellets were suspended in water. The final purification of oligonucleotides was

performed on an Agilent Prostar System (Agilent, Santa Clara, CA) equipped with a

Hamilton HxSil C18 column (150x21.2). The pure oligonucleotides were collected,

desalted by size-exclusion chromatography using a Sephadex G25 and lyophilized. LC-

MS analysis of oligonucleotides .The identity of oligonucleotides was established by LC-

MS analysis on an Agilent 6530 accuratemass Q-TOF LC/MS (Agilent technologies,

Santa Clara, CA).

The purified strands were duplexed and duplex formation and purify confirmed by gel.

"Living" GFP+(%of total cells in upper left quadrant)

Dying GFP+(% of total cells in upper right quadrant)

"Living" GFP-(% of total cells in lower left quadrant)

"dead" cells(% of total cells in lower right quadrant)

Control48 h 74.3 % 0.04 % 24.3 % 1.28 %Control72 h 40.9 % 0.20 % 58.0 % 0.82 %Lipofection 48 h 12.2 % 0.00 % 83.9 % 3.89 %Lipofection 72 h 14.8 % 0.00 % 84.2% 0.95 %

a a

"

A Control48h Lipofection 48h

·._Q1

mNP48h1-UL4.8%

FIGURE S-5

Ethidium fluorescence

8 -a

,-----------------,Number of living GFP+ cells

- - - -a

..,-,---------------,

Number of living GFP+ cellsControl vs Lipofection

- -

-,--------------,Number of living GFP+ cells

Control vs mNPs

0::

"0

..1 .;2 .;3 ..4 w5 ,p ..7FL1-A

GFP fluorescence

c Control72h

0:: "0 u

0::

"0:J

..1 .;2 .;3 ..4 w5 ,p ..7FL1-A

GFP fluorescence

Lipofection 72h

...

.;2 .;3 w5 ,p 2FL1-A

GFP fluorescence

mNP 72h

Ethidium fluorescence2

Ethidium fluorescenceJ2

Ethidium fluorescencea-,----- - ---===----- - --, -,-----------------,

D Number of living GFP+ cells

--

Number of living GFP+ cellsControl vs Lipofection

Number of living GFP+ cellsControl vs mNPs

- -0:::::>0u

a'<>

0::

0u

..1 .;2 .;3 ..4 w5 ,pFL1-A

GFP fluorescence

E

.;2 .;3 ..4 w5 ,p ..7FL1-A

GFP fluorescence

..1 .;2 .;3 ..4 w5 ,p ..7.2FL1-A

GFP fluorescence

"Living" GFP+(%of total cells in upper left quadrant)

Dying GFP+(% of total cells in upper right quadrant)

"Living" GFP-(% of total cells in lower left quadrant)

"dead" cells(% of total cells in lower right quadrant)

Control48 h 74.3 % 0.04 % 24.3 % 1.28 %Control72 h 40.9 % 0.20 % 58.0 % 0.82 %Lipofection 48 h 12.2 % 0.00 % 83.9 % 3.89 %Lipofection 72 h 14.8 % 0.00 % 84.2% 0.95 %


Legend for Figure S-5

FACS was performed to enumerate GFP+ and ethidium+ cells in eGFP expressing

NIHT3 cells. The cell cultures were incubated for 48 or 72 h with either mNPs or

lipofectamine bearing anti-GFP siRNA. Ethidium staining was used to identify dying or

dead cells. Panels from A and C present FACS data as dot plots showing GFP

silencing after 48 and 72 h, respectively. Dot plots indicate the number of cells

(expressed as percentage of total cells counted) that are GFP+ and ethidium+ or both.

The X-axis indicates ethidium fluorescence intensity and the Y-axis shows GFP

fluorescence intensity. In each dot plot panel, cells in the upper left quadrant are GFP+

cells that have no ethidium staining (e.g. living GFP+ cells). The left lower quadrants

show living cells that do not express GFP above the threshold. Notice that under

control conditions 24% of cells have very low GFP fluorescence at 48 h (Panel A) and

58% at 72 h (Panel C). The right upper quadrants indicate GFP+ cells that also are

stained with ethidium (dying GFP+ cells). The right lower quadrant shows ethidum+

cells that do not express GFP (dead cells). Panels B and D show the data as cell

counts (Y-axis) plotted against GFP fluorescence intensity. Panel E summarizes the

data in numerical form, indicating that mNP cytotoxicity was not significantly different

from that elicited by lipofection. Indeed it was slightly less with mNP than with

lipofection after 72 h.

% e

nhan

cem

ent

% e

nhan

cem

ent

% e

nhan

cem

ent

% e

nhan

cem

ent

125

100

75

50

25

Olfact*ory Bulb

*

**

Group 1Group 2

100

75

50

25

Hippocampus

*

FIGURE 6Group 1Group 2

0

100

75

50

0 24 48Time (hrs)

Cerebral Cortex

*

Group 1Group 2

0

100

75

50

0 10 20 30 40 50Time (hrs)

Corpus Striatum

*

Group 1Group 2

*25 25

00 10 20 30 40 50

Time (hrs)

00 10 20 30 40 50

Time (hrs)



MRI experiment conducted with earlier coil and protocols before upgrading to the

MRI system described in methods and reported in Fig 3. Mean manganese

signal (T1-weighted MR) was measured in 4 brain regions (N=6 mice per group)

at 24 and 48 hrs. Group 1 (circles) received nanoparticles containing high

concentrations of Mn (58 µM with an estimated 470 Mn molecules

per nanoparticle) and Group 2 (squares) received nanoparticles with lower

concentrations of Mn (29 µM with an estimated 235 Mn molecules

per nanoparticle). One-way ANOVA was performed separately for low and high

concentraton Mn NPs in each of 4 brain regions using Matlab Statistics Toolbox

(Mathworks, Inc., Natick, MA) * p <0.05

FIGURE S-7



GFP is not expressed universally in all neurons from Tg green mice utilized in this study.

As shown here, GFP expression is observed in sub-populations of neurons at variable

intensities of fluorescence. Therefore measurements of GFP fluoresence distribution

and intensity is not useful in detecting GFP silencing in the short-term. Panels A to I

show GFP+ neurons in Tg GFP mice demonstrating that these mice only express GFP

at various fluorescence intensities in sub-populations of neurons: A) Olfactory bulb (OB),

scale bar= 200 µm; B) OB glomerular layer, scale bar= 20 µm; C) OB neurons scale

bar= 10 µm; D) Frontal cerebral cortex neurons (CTX), scale bar= 50

µm; E) CTX neurons, scale bar= 20 µm; F) Hippocamal dentate gyrus neurons of sub-

granular layer, scale bar= 20 µm; G) Striatal neurons, scale bar= 20 µm; H) striatal

neurons, scale bar= 20 µm; I) Purkinje neurons of cerebellum, scale bar= 50 µm.

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