Supplementary Information

Enhanced tendon restoration effects of anti-inflammatory lactoferrin-immobilized

heparin-polymeric nanoparticles on Achilles tendinitis rat model

Hong Joon Choia,b,1, Somang Choic,1, Jae Gyoon Kimd,1, Mi Hyun Songe, Kyu-Sik Shime,

Youn-Mook Limf, Hak-Jun Kime,*, Kyeongsoon Parkg,*, Sung Eun Kime,*

aDepartment of Orthopedic Surgery, College of Medicine Korea University, Anam-dong, Seongbuk-

gu, Seoul 02841, KoreabYonsei gunwoo Hospital, #1814, Nambusunhwan-ro, Gwanak-gu, Seoul 08787, Korea cDepartment of Biomedical Science, College of Medicine Korea University, Anam-dong, Seongbuk-

gu, Seoul 02841, KoreadDepartment of Orthopedic Surgery, Korea University, College of Medicine, Korea University Ansan

Hospital 123, Jeokgeum-ro, Danwon-gu, Ansan-si, Gyeonggi-do, 15355, KoreaeDepartment of Orthopedic Surgery and Rare Diseases Institute, Korea University Guro Hospital, #80,

Guro-dong, Guro-gu, Seoul 08308, KoreafAdvanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-

dong, Jeongeup-si, Jeollabuk-do 56212, Republic of KoreagDepartment of Systems Biotechnology, Chung-Ang University, Anseong, Gyeonggi 17546, Korea

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Materials

PLGA (50:50; Resomer RG505) was supplied by Boehringer Ingelheim (Ingelheim,

Germany). Poly(vinyl alcohol) (PVA, molecular weight: 13,000–23,000, 98% hydrolyzed),

DOPA, dichloromethane, dimethyl sulfoxide, and LF were purchased from Sigma-Aldrich

(St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum

(FBS), phosphate-buffered saline (PBS), antibiotics, and collagenase type I were purchased

from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Heparin sodium (molecular

weight: 12,000–15,000 g/mol) was obtained from Acrose Organics (NJ, USA). A cell

counting kit (CCK)-8 was purchased from Dojindo Molecular Technologies, Inc. (Rockville,

MD, USA).

Real-time PCR condition

Each PCR run was followed by a melt analysis to confirm the specificity of reaction products.

Real-time PCR was performed by initial denaturation at 95 for 10 min and 50 cycles of℃

denaturation at 95 for 15 sec, 50 cycles of annealing at 58 for 1 min, and an extension℃ ℃

step at 95 for 15 sec, at 60 for 1 min, at 95 for 15 sec, and at 60 for 15sec.℃ ℃ ℃ ℃

Amplification and melting curves of each product were shown in Fig. S1, S2, and S3.

Table S1. RT-PCR primer sequences of the genes related to pro-inflammatory factors

Gene Primer (forward) 5 3 Primer (reverse)

COX-2 CAG CCA TAC TAT GCC TCG GA GGA TGT CTT GCT CGT CGT TC

IL-1β CCA CCT CCA GGG ACA GGA TA AAC ACG CAG GAC AGG TAC AG

MMP-3 ACC TGT CCC TCC AGA ACC TG AAC TTC ATA TGC GGC ATC CA

2

MMP-13 AAG GAG CAT GGC GAC TTC TA GGT CCT TGG AGT GGT CAA GA

IL-6 CCG TTT CTA CCT GGA GTT TG GTT TGC CGA GTA GAC CTC AT

TNF-α CTC CCA GAA AAG CAA GCA AC CGA GCA GGA ATG AGA AGA GG

Table S2. RT-PCR primer sequences of the genes related to anti-inflammatory factors

Gene Primer (forward) 5 3 Primer (reverse)

IL-4 ACA GGA GAA GGG ACG CCA T GAA GCC CTA CAG ACG AGC TCA

IL-10 GGT TGC CAA GCC TTA TCG GA ACC TGC TCC ACT GCC TTG CT

Table S3. RT-PCR primer sequences of the genes related to tenogenic markers

Gene Primer (forward) 5 3 Primer (reverse)

COL1A1 CCC TGG AAA GAA TGG AGA TG CCA CTG AAA CCT CTG TGT CC

DCN CCA GAA AAA ATG CCC AAA ACT C TCG CAC TTT GGT GAT CTC ATT C

TNC GGG TCC TCA AGA AAG TCA TCC G CTG ACT CCA GAT CCA CCG AAC

3

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Fig. S1. Amplification and melting curves of pro-inflammatory factors. (a) COX-2, (b) IL-1β,

(c) MMP-3, (d) MMP-13, (e) IL-6, and (f) TNF-α in Real-time PCR analysis.

Fig. S2. Amplification and melting curves of anti-inflammatory factors. (a) IL-4 and (b) IL-

10.

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Fig. S3. Amplification and melting curves of tenogenic markers. (a) COL1A1, (b) DCN, and

(c) TNC.

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Fig. S4. FT-IR spectra of (a) heparin, (b) lactoferrin, (c) PLGA, (d) Hep-PGLA, and (e)

LF(10 g)/Hep-PLGA NPs.

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