Supplementary Materials and Methods
Materials for Polymer Synthesis. Chemicals and all materials were supplied by
Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Cyano-4-
(ethylsulfanylthiocarbonyl)sulfanyl pentanoic acid (ECT) was synthesized as described previously (20). Biotin-dPEG TM
3
-NH
3
+ TFA was obtained from Quanta Biodesign
(Powell, Ohio). 2,2′-Azobis(4-methoxy-2.4-dimethyl valeronitrile) (V-70) was obtained from Wako Chemicals (Richmond, VA).
1
Synthesis of N-hydroxy succinimide activated ECT. A solution of ECT (2 g, 7.59 mmol) and NHydroxysuccinimide (NHS) (2.6206 g, 22.77 mmol) in chloroform (200 mL) was purged by nitrogen for 1 hour and cooled over an ice bath. N , N
′-
Dicyclohexylcarbodiimide (6.2642 g, 30.36 mmol) was slowly added while vigorously stirring the mixture. The reaction remained in the ice bath for 1 hour, then removed and allowed to react at room temperature for 22 hours. The reaction mixture was filtered and the organic solvent was removed by vacuum. The NHS activated ECT was redissolved in chloroform (200 mL) and propargylamine (0.1808 g, 3.28 mmol) was then slowly added. The reaction mixture was stirred at room temperature for 18 hours.
Chloroform (100 mL) was added and the mixture was extracted with an aqueous solution of hydrochloric acid (1/10 v/v, 5 x 100 mL), 10 wt% sodium hydroxide (5 x 100 mL) and ddH
2
O water (2 x 100 mL). The chloroform solution was dried over magnesium sulfate. After solvent removal by vacuum, three successive column chromatographies (Silica gel 60) were performed (ethyl acetate/hexanes (75%/25%),

ethyl acetate (100%), chloroform/methanol (98%/2%). After drying under vacuum, a viscous orange-red oil was obtained (yield 92%).
1 H NMR 300MHz (CDCl
3
, RT, ppm)
(see structure below for proton assignment): 1.35 (t, 3 H, CH
3
); 1.89 (s, 3 H, CH3); 2.2-
2.6 (m, 5 H, CH2CH2, CH); 3.34 (q, 2 H, CH2); 4.00 (m, 2 H, CH2); 5.93 (b,1 H, NH).
2
Synthesis of BIOTIN-PEO
3
-ECT. The synthesis of the biotinylated RAFT chain transfer agent was accomplished according to a related previously described procedure (22-23).
Briefly Biotin-dPEG TM
3
-NH
3
+ TFA - (2.0 g, 3.567 mmol) and triethylamine (720 mg, 7.1 mmol) in 200 mL chloroform was added dropwise in three equal portions 20 minutes apart to a stirred solution of N-hydroxy succinimide activated ECT (1.41 g, 3.92 mmol) in 350 mL chloroform. The reaction was allowed to stir at room temperature for 18 hours. The chloroform was then removed by rotary evaporation and the product isolated by column chromatography (silica gel stationary phase, 9:1 v/v CH
2
Cl
2
:CH
3
OH).
1 H NMR: (CDCl
3
) δ 1.38 t (SCH
2
C H
3
); δ 1.93 s (CCNC H
3
); δ 2.3–2.65 m (C H
2
C H
2
); δ
3.35 q (SC H
2
CH
3
convoluted). 4.35-4.6 (m, CHSC H
2
); δ 2.75-2.9 (dd, C
H C H
); δ 5.15-
5.79 (S, N H CON H ); δ 2.85-291 (d, CHC H CH); δ 2.72-2.74 (dd, CHC H CH
2
); δ 4.28-4.54
(SC H
2
CH); δ 3.14 (dt, C H
2
CHS ); δ 2.23 (t, COC H
2
CH
2
CH
2
CH
2
); δ 1.44 (dt, C H
2
CHS); δ
1.68 (overlapping multiplet, COCH
2
C H
2
CH
2
C H
2
); δ 3.5-3.6 (C
H
2
C H
2
C H
2
(OC H
2
C H
2
)
3
, multiplets).
HABA assay.
A modification of the HABA (4-hydroxyazobenzene2’carboxylic acid) assay was used to measure available biotin sites on polymeric micelles. HABA dye

bound to streptavidin (HABA-SA) absorbs light at 500 nm wavelength (A
500
). Biotin competitively displaces HABA from HABA-SA resulting in reduced A
500
. A competitive
3
HABA assay was used whereby mAb-SA bearing polymeric micelles were formed by incubating polymer with siRNA 30 minutes prior to addition of mAb-SA at varying concentrations. This mixture was then added to HABA-SA solution containing 265

M
HABA and 10.9

M streptavidin. The solution was vortexed briefly then incubated for
15 minutes. The A
500 was measured and absorbance difference was calculated by subtracting A
500 of polyplex solution from A
500 of HABA-SA solution. Unbound biotin sites on polymeric micelles displace HABA from HABA-SA resulting in reduced A
500 reading. Conversely, excess unbound mAb-SA binds to free HABA and is measured by increased A
500
.
Complexation of siRNA by polymer.
Binding of siRNA by polymer was shown via agarose gel electrophoresis demonstrating retardation of siRNA migration after incubation with polymer. Briefly, 200 ng of siRNA was incubated either alone or with increasing concentrations of polymer with or without mAb-SA conjugate for 30 minutes at room temperature. Complexes were loaded into a 2% agarose gel and run for 1.5 hours at 60V in 1X TAE buffer, pH 7.4 and subsequently stained in Sybr Green II RNA
Gel Stain (Invitrogen, 1:5000 dilution in TAE buffer).
Binding of mAb-SA conjugate to polymer.
Protein electrophoresis under nondenaturing conditions was used to evaluate binding of mAb-SA conjugate to polymer.

8.4 picomoles of HD39-SA conjugate was incubated for 30 minutes in the presence of
211 picomoles of polymer alone or polymer complexed to 20 picomoles of siRNA.
Complexes were diluted 1:1 with 2X Tris Glycine Native Sample Buffer then loaded into a 3-8% NuPage Tris Acetate gel (Invitrogen) and run for 1 hour at 150V in 1X Tris
4
Glycine Native Running Buffer (Invitrogen). The gel was subsequently stained with
Coomassie Blue protein stain.
Polymeric micelle internalization and microscopy in HeLa-R cells. HeLa-R cells were plated in 12 well plates at 50,000 cells per well in 500
 l of media the day before transfection. Media was changed on the day of transfection. Complexes were formed by incubating non-biotinylated polymer with Silencer Cy3-labeled Negative control siRNA #1 (AM4621; Ambion) at a 5:1 polymer to siRNA molar ratio and added to HeLa-
R cells for a final siRNA dose of 100 nM. Treated cells were trypsinized after 24 hours of treatment then cytospun on slides, dried, then fixed with 10% neutral buffered formalin and rinsed in PBS prior to being coverslipped with Prolong Gold antifade reagent with DAPI (Invitrogen). Random fields were imaged using a Nikon Eclipse
E800 wide-field fluorescence microscope fitted with 100x/1.30 PlanFluor numerical aperture objective. Images were collected with MetaMorph software (Molecular
Devices). Scale bars were added with public domain software ImageJ.
Cell viability assessment.
HeLaR CD22 cells were plated the day before treatment in white 96-well assay plates with clear bottoms at a density of 3,000 cells per well in 100

 l of culture media. On the day of treatment, the media was changed and cells treated in quadruplicate with polymeric micelles at 15 nM siRNA final dose in 100
 l volume.
5
Cells were harvested 24 hours after treatment and luminescence readings obtained according to the CellTiter-
Glo kit manufacturer’s protocol (Promega). Briefly, the plate was equilibrated to room temperature for 30 minutes prior to the addition of 100
 l of
CellTiter-Glo reagent to each well. Plates were protected from light then mixed thoroughly to lyse cells. After 10 minute incubation at room temperature, luminescence was measured using a Centro LB 960 microplate luminometer (Berthold Technologies).
Transfections and qRT-PCR for NAC1 and Mcl-1.
Transfections for NAC1 and Mcl-1 were performed as described for GAPD siRNA in the Methods section. Sequences for the siRNAs used are as follows: Myeloid Cell Leukemia sequence 1, Mcl-1 (sense strand 5’-CCAGUAUACUUCUUAGAAATT-3’; Ambion); Nucleus Accumbens
A ssociated 1, NAC1 (sense strand 5’-CGAGAAAUUGCACAACCGATT-3’; Ambion).
RNA was extracted using the RNeasy Mini Kit (Qiagen). Quantitative RT-PCR was performed and analyzed as described in the Methods section. FAM-labeled probe and primers were obtained as 20X primer-probe mixes from Applied Biosystems: Mcl-1
(Catalog#Hs00172036_m1), NAC1 (Catalog#Hs00369413_m1).

6
References
1. Jemal A, Siegel R, Xu J, Ward E (2010). Cancer Statistics, 2010. CA Cancer J
Clin 60 : 277-300.
2. Tiemann K, Rossi JJ (2009). RNAi-based therapeutics-current status, challenges and prospects. EMBO Mol Med 1 : 142-51.
3. Phalon C, Rao DD, Nemunaitis J (2010). Potential use of RNA interference in cancer therapy. Expert Rev Mol Med 12 :e26.
4. Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery (2009). Nat Rev Drug Discov 8 : 129-38.
5. Dominska M, Dykxhoorn DM (2010). Breaking down the barriers: siRNA delivery and endosome escape. J Cell Sci 123 : 1183-9.
6. Ikeda Y, Taira K (2006). Ligand-targeted delivery of therapeutic siRNA. Pharm
Res 23 : 1631-40.
7. Haas KM, Sen S, Sanford IG, Miller AS, Poe JC, Tedder TF (2006). CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo. J Immunol 177 :
3063-73.
8. Vitetta ES, Stone M, Amlot P, et al (1991). Phase I immunotoxin trial in patients with B-cell lymphoma. Cancer Res 51 : 4052-8.
9. Coleman M, Goldenberg DM, Siegel AB, et al (2003). Epratuzumab: targeting Bcell malignancies through CD22. Clin Cancer Res 9 : 3991S-4S.
10. Shan D, Press OW (1995). Constitutive endocytosis and degradation of CD22 by human B cells. J Immunol 154 : 4466-75.

7
11. Press OW, Farr AG, Borroz KI, Anderson SK, Martin PJ (1989). Endocytosis and degradation of monoclonal antibodies targeting human B-cell malignancies. Cancer Res
49 : 4906-12.
12. Carnahan J, Wang P, Kendall R, et al (2003) .
Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties. Clin Cancer
Res 9 : 3982S-90S.
13. Du X, Beers R, Fitzgerald DJ, Pastan I (2008). Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity. Cancer Res
68 : 6300-5.
14. Leonard JP, Schuster SJ, Emmanouilides C, et al (2008). Durable complete responses from therapy with combined epratuzumab and rituximab: final results from an international multicenter, phase 2 study in recurrent, indolent, non-Hodgkin lymphoma.
Cancer 113 : 2714-23.
15. Leonard JP, Coleman M, Ketas JC, et al (2004). Epratuzumab, a humanized anti-CD22 antibody, in aggressive non-Hodgkin's lymphoma: phase I/II clinical trial results. Clin Cancer Res 10 : 5327-34.
16. O'Donnell RT, Martin SM, Ma Y, Zamboni WC, Tuscano JM (2010). Development and characterization of CD22-targeted pegylated-liposomal doxorubicin (IL-PLD). Invest
New Drugs 28 : 260-7.
17. Mansfield E, Amlot P, Pastan I, FitzGerald DJ (1997). Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors. Blood
90 : 2020-6.

18. Bogner C, Dechow T, Ringshausen I, et al (2010). Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression. Br J
Haematol 148 : 99-109.
19. Ghetie MA, May RD, Till M, et al (1988). Evaluation of ricin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagents for in vivo therapy. Cancer Res 48 : 2610-7.
20. Convertine AJ, Benoit DS, Duvall CL, Hoffman AS, Stayton PS (2009).
Development of a novel endosomolytic diblock copolymer for siRNA delivery. J Control
Release 133 : 221-9.
8
21. Convertine AJ, Diab C, Prieve M, et al (2010). pH-Responsive Polymeric Micelle
Carriers for siRNA Drugs. Biomacromolecules ; in press.
22. Bathfield M, D'Agosto F, Spitz R, Charreyre MT, Delair T (2006). Versatile precursors of functional RAFT agents. Application to the synthesis of bio-related endfunctionalized polymers. J Am Chem Soc 128 : 2546-7.
23. Hong C, Pan C (2006). Direct synthesis of biotinylated stimuli-responsive polymer and diblock copolymer by RAFT polymerization using biotinylated trithiocarbonate as RAFT agent. Macromolecules 39 : 3517-24.
24. Pagel JM, Pantelias A, Hedin N, et al (2007). Evaluation of CD20, CD22, and
HLA-DR targeting for radioimmunotherapy of B-cell lymphomas. Cancer Res 67 : 5921-
8.

9
25. Dorken B, Moldenhauer G, Pezzutto A, et al (1986). HD39 (B3), a B lineagerestricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes. J Immunol 136 : 4470-9.
26. Hylarides MD, Mallett RW, Meyer DL (2001). A robust method for the preparation and purification of antibody/streptavidin conjugates. Bioconjug Chem 12 : 421-7.
27. Pagel JM, Hedin N, Subbiah K, et al (2003). Comparison of anti-CD20 and anti-
CD45 antibodies for conventional and pretargeted radioimmunotherapy of B-cell lymphomas. Blood 101 : 2340-8.
28. James SE, Greenberg PD, Jensen MC, et al (2008). Antigen sensitivity of CD22specific chimeric TCR is modulated by target epitope distance from the cell membrane.
J Immunol 180 : 7028-38.
29. Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E (1994). The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J Biol Chem 269 : 12918-24.
30. Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 : 402-8.
31. Davis ME, Zuckerman JE, Choi CH, et al (2010). Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464 : 1067-70.
32. Fang J, Nakamura H, Maeda H (2010). The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev May 2.

10
33. Matsumura Y, Maeda H (1986). A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 46 : 6387-92.
34. Xia CF, Boado RJ, Pardridge WM (2009). Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology. Mol Pharm 6 : 747-51.
35. Xia CF, Zhang Y, Boado RJ, Pardridge WM (2007). Intravenous siRNA of brain cancer with receptor targeting and avidin-biotin technology. Pharm Res 24 : 2309-16.
36. Chu TC, Twu KY, Ellington AD, Levy M (2006). Aptamer mediated siRNA delivery. Nucleic Acids Res 34 :e73.
37. Conner SD, Schmid SL (2003). Regulated portals of entry into the cell. Nature
422 : 37-44.
38. Goldmacher VS, Tinnel NL, Nelson BC (1986). Evidence that pinocytosis in lymphoid cells has a low capacity. J Cell Biol 102 : 1312-9.
39. Dearling JL, Park EJ, Dunning P, et al (2010). Detection of intestinal inflammation by MicroPET imaging using a (64)Cu-labeled anti-beta(7) integrin antibody. Inflamm Bowel Dis 16 : 1458-66.
40. Merkerova M, Klamova H, Brdicka R, Bruchova H (2007). Targeting of gene expression by siRNA in CML primary cells. Mol Biol Rep 34 : 27-33.
41. Seiffert M, Stilgenbauer S, Dohner H, Lichter P (2007). Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids. Leukemia 21 : 1977-
83.

11
42. Kumar P, Ban HS, Kim SS, et al (2008). T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134 : 577-86.
43. Song E, Zhu P, Lee SK, et al (2005). Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 23 : 709-17.
44. Peer D, Zhu P, Carman CV, Lieberman J, Shimaoka M (2007). Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc Natl Acad Sci USA 104 : 4095-100.
45. Peer D, Park EJ, Morishita Y, Carman CV, Shimaoka M (2008). Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target.
Science 319 : 627-30.
46. Kim SS, Peer D, Kumar P, et al (2010). RNAi-mediated CCR5 silencing by LFA-
1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 18 : 370-6.
47. Meyer DL, Schultz J, Lin YK, et al (2001). Reduced antibody response to streptavidin through site-directed mutagenesis. Protein Sci 10 : 491-503.
48. Chinol M, Casalini P, Maggiolo M, et al (1998). Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Brit J
Cancer 78 : 189-97.
49. Lasham A, Herbert M, Wallant NC, Patel R, et al (2010). A rapid and sensitive method to detect siRNAmediated mRNA cleavage in vivo using 5’ RACE and a molecular beacon probe. Nucleic Acids Res 38 : e19.