PUBLICATIONS

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2020 year (updated, 2020.05)

1. Shin HJ, Park H, Shin N, Kwon HH, Yin Y, Hwang JA, Kim SI, Kim SR, Kim S, Joo Y, Kim Y, Kim J, Beom J, Kim DW*.

p47phox siRNA-Loaded PLGA Nanoparticles Suppress ROS/Oxidative Stress-Induced Chondrocyte Damage in Osteoarthritis. 

Polymers (Basel). 2020 Feb 13;12(2). pii: E443. 

doi: 10.3390/polym12020443. PubMed PMID: 32069893.
https://www.mdpi.com/2073-4360/12/2/443

3: Shin HJ, Park H, Shin N, Shin J, Gwon DH, Kwon HH, Yin Y, Hwang JA, Hong J, Heo JY, Kim CS, Joo Y, Kim Y, Kim J, Beom J, Kim DW*.
p66shc siRNA Nanoparticles Ameliorate Chondrocytic Mitochondrial Dysfunction in Osteoarthritis.
Int J Nanomedicine. 2020 Apr 8;15:2379-2390.
doi: 10.2147/IJN.S234198. eCollection 2020. PubMed PMID: 32308389; PubMed Central PMCID: PMC7152540.

4: Phạm TL, Yin Y, Kwon HH, Shin N, Kim SI, Park H, Shin J, Shin HJ, Hwang JA, Song HJ, Kim SR, Lee JH, Hwang PTJ, Jun HW, Kim DW*.
miRNA 146a-5p-loaded poly(d,l-lactic-co-glycolic acid) nanoparticles impair pain behaviors by inhibiting multiple inflammatory pathways in microglia.
Nanomedicine (Lond). 2020 Apr 15.
doi: 10.2217/nnm-2019-0462. [Epub ahead of print] PubMed PMID: 32292108.

5. Shin N, Shin HJ, Yi Y, Beom J, Lee W, Lee CH, Kim DW*
p66shc siRNA-Encapsulated PLGA Nanoparticles Ameliorate Neuropathic Pain Following Spinal Nerve Ligation

Polymers 2020, 12(5), 1014; 

https://doi.org/10.3390/polym12051014

                                      




6. CX3CR1-Targeted PLGA Nanoparticles Reduce Microglia Activation and Pain Behavior in Rats with Spinal Nerve Ligation.
Noh C, Shin HJ, Lee S, Kim SI, Kim YH, Lee WH, Kim DW, Lee SY, Ko YK.

Int J Mol Sci. 2020 May 14;21(10):E3469. 

doi: 10.3390/ijms21103469.PMID: 32423102


7. Phạm TL, Kim DW*.
Poly(lactic-co-glycolic acid) nanomaterial-based treatment options for pain management: a review.
Nanomedicine (Lond). 2020 Aug 6.
doi: 10.2217/nnm-2020-0114. Epub ahead of print. PMID: 32757701.

2019 year

1. Shin J, Yin Y, Kim DK, Lee SY, Lee W, Kang JW, Kim DW*, Hong J.
Foxp3 plasmid-encapsulated PLGA nanoparticles attenuate pain behavior in rats with spinal nerve ligation.
Nanomedicine. 2019 Mar 8. pii: S1549-9634(19)30054-1.
doi: 10.1016/j.nano.2019.02.023. [Epub ahead of print] PubMed PMID: 30858084.
https://doi.org/10.1016/j.nano.2019.02.023

Figure 1. Experimental scheme of this study. Neuropathic pain was induced in rats with lumbar 5 (L5) spinal nerve ligation (SNL). Negative control or Foxp3 plasmid (pFoxp3)-encapsulated poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (NC NPs and Foxp3 NPs, respectively) were then delivered intrathecally into the spinal subarachnoid space of test animals using a Hamilton syringe with a 26.5 G needle. Afterwards, the animals were subjected to behavioral tests using von Frey filaments to evaluate the analgesic effect of Foxp3 NPs on SNL-induced neuropathic pain.

Figure 2. Foxp3 expression in rats with SNL-induced neuropathic pain. (A) On days 3 and 7 post-SNL operation, total RNA was isolated from L4–L5 segments of the spinal cord ipsilateral dorsal horn and utilized for cDNA synthesis. The mRNA level of Foxp3 was analyzed by quantitative real-time PCR (qPCR). A sham group was used as the control. Data are presented as mean ± standard error of the mean (SEM) (one-way analysis of variance [ANOVA] with Turkey’s post-hoc test; ***P < 0.001 and *P < 0.01 vs. Sham; n = 4–6 per group). POD = post-operative day. (B) On days 3 and 7 post-SNL surgery, L5 spinal sections were prepared and incubated with anti-Foxp3 antibodies. Arrowheads indicate Foxp3-positive cells. Scale bar = 50 μm (left), 10 μm (right).

Figure 3. Foxp3 overexpression suppresses the mRNA expression of proinflammatory genes in LPS-stimulated BV2 cells. (A) The pFoxp3 was constructed by subcloning of the coding sequence (1,287 base pairs) of mouse Foxp3 into the negative control plasmid (pNC; left). The constructs were confirmed by the mCherry expression under a fluorescent microscope (middle), and the Foxp3 expression (right) by immunoblot analysis in HEK293T cells transfected with pNC or pFoxp3. β-actin was used as the loading control (right). Data are expressed as mean ± SEM (unpaired Student’s t-test; ***P < 0.001 vs. pNC-transfected cells). (B) pNC or pFoxp3 were transfected into BV2 cells for 2 days. Then, the mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 in the absence or presence of lipopolysaccharide (LPS) (100 ng/ml) were assessed for 3 hours by qPCR. Data are expressed as mean ± SEM (one-way ANOVA with Turkey’s post-hoc test; ***P < 0.001 and **P < 0.05 vs. pNC + LPS).

Figure 4. Physical characterization and cytotoxicity of the plasmid-encapsulated PLGA NPs. (A) Plasmid-encapsulated PLGA NPs were prepared by briefly sonicating the mixture of PLGA and plasmid, and then examined for particle size and zeta potential using a Zetasizer Nano ZS and scanning electron microscopy. Scale bar = 500 nm. (B) BV2 cells were incubated with NPs (0–200 μg/mL) for 24 h. Their cytotoxicity was then investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are expressed as mean ± SEM (one-way ANOVA test; n.s., not significant).

Figure 5. Foxp3 NPs efficiently inhibit the transcription of proinflammatory mediators in LPS-stimulated BV2 cells. (A) HEK293T cells were treated with NC NPs or Foxp3 NPs for 2 days. The expression levels were then determined by mCherry under a fluorescent microscope (left) and Foxp3 on western blot (right). (B) NC NPs or Foxp3 NPs (100 μg/mL) were added to BV2 cells for 2 days. Later, the mRNA expression levels of proinflammatory mediators, such as TNF-α, IL-1𝛽 and IL-6, after 3-h LPS treatment (100 ng/ml), were measured by qPCR. Data are expressed as mean ± SEM (one-way ANOVA with Turkey’s post-hoc test; **P < 0.01 and *P < 0.05 vs. NC NPs + LPS). 

Figure 6. Foxp3 NPs mainly localize in the spinal microglia of SNL-induced rats. (A) Foxp3 NPs were intrathecally administered to SNL-induced rats. On day 4 post-injection, L5 fragments of the spinal cord were isolated, sectioned and used for immunostaining with anti-Foxp3 antibodies to examine the expression of Foxp3 in Foxp3 NPs-localized cells. Arrowheads indicate mCherry⁺/Foxp3⁺- positive cells. Scale bar = 20 μm (left), 10 μm (right). (B) The same spinal tissues described above were also immunostained with anti-Iba1 (microglial marker), GFAP (astrocytic marker) and NeuN (neuronal marker) antibodies. Arrowheads indicate mCherry⁺/Iba1⁺-, mCherry⁺/GFAP⁺- and mCherry⁺/Neuron⁺-positive cells. Scale bar = 20 μm. Data are expressed as mean ± SEM (one-way ANOVA with Turkey’s post-hoc test; ***P < 0.001 vs. mCherry⁺/Iba1⁺- positive cells). 

Figure 7. Foxp3 NPs notably attenuate the mechanical hypersensitivity of rats with SNL by reducing spinal microglia activation. (A) After environmental adaptation for 1 week, rats insensitive to the von Frey filaments test (> 10 g filament) were used for SNL surgery. On day 3 post-surgery, sensitive rats (< 4 g filament) were randomly divided into two groups (NC NPs and Foxp3 NPs) and intrathecally injected with two NPs. Later, several tests for pain behavior and gene expression were conducted at the indicated-time points. (B) After NPs injection, von Frey filaments tests were performed daily from day 5 to 10 post-surgery. Data are presented as mean ± SEM (two-way ANOVA with Bonferroni’s post-hoc test; ***P < 0.001 vs. NC NPs on day 7; n = 5–6). (C) L5 spinal sections from the sham group, POD3, and POD7, were prepared and used for immunostaining with anti-Iba1 antibodies. Dotted lines indicate the ipsilateral side of the spinal dorsal horn. Scale bar = 50 μm. Data are presented as mean ± SEM (two-way ANOVA with Bonferroni’s post-hoc test; ***P < 0.001 vs. NC NPs on day 7; n = 3–4). POD, post-operative day.

Figure 8. Foxp3 NPs modulate the expression of neuropathic pain-related genes in the spinal dorsal horn of SNL-induced rats. (A) On day 4 post-injection of NPs, total RNA was isolated from the L4–L5 segment (0.7 cm) of the ipsilateral spinal dorsal horn and used for cDNA synthesis. Later, mRNA levels of TNF-α, IL-1β, IL-6 and cyclooxygenase (COX)-2 were determined by qPCR. Data are presented as mean ± SEM (one-way ANOVA with Turkey’s post-hoc test; ***P < 0.001 and **P < 0.01 vs. NC NPs on day 7; n = 3–5 per group). (B) The same sections used in Figure 6C were double-immunostained with anti-Iba1 and anti- inducible nitric oxide synthase (iNOS) and anti-arginase 1 (Arg1) antibodies. Arrowheads indicate iNOS⁺/Iba1⁺- or Arg1⁺/Iba1⁺- positive cells. Scale bar = 50 μm (left), 10 μm (right). Data are presented as mean ± SEM (one-way ANOVA with Turkey’s post-hoc test; ***P < 0.001, **P < 0.01, and *P < 0.05 vs. NC NPs; n = 6–8 per group).

2. Shin J, Yin Y, Park H, Park S, Triantafillu UL, Kim Y, Kim SR, Lee SY, Kim DK, Hong J, Kim DW*.
p38 siRNA-encapsulated PLGA nanoparticles alleviate neuropathic pain behavior in rats by inhibiting microglia activation.
Nanomedicine (Lond). 2018 Jul;13(13):1607-1621.
doi: 10.2217/nnm-2018-0054. PubMed PMID: 30028250.
https://goo.gl/rnqfH6

Figure 1. Experimental scheme of this study. Neuropathic pain in rats was induced by L5 spinal nerve ligation. Scrambled siRNA- or p38 siRNA-encapsulated PLGA nanoparticles were then delivered intrathecally into the subarachnoid space using a Hamilton syringe. Rats were then subjected to behavioral tests using von Frey filaments to evaluate the effect of p38siRNA PLGA nanoparticles on neuropathic pain. 

Figure 2. p38 siRNA inhibits p38 expression in BV2 cells and reduces expression of proinflammatory mediators in LPS-stimulated BV2 cells

(A) p38 siRNA (10, 20, and 50 nM) or scrambled siRNA (sc siRNA, 20 nM) were transfected into BV2 cells with RNAiMAX reagents. After 2 d, p38 mRNA levels were assessed by quantitative real-time PCR (qPCR). (B) sc siRNA (10 nM) or p38 siRNA (10 nM) were transfected into BV2 cells for 2 d. The expression of p38 or p-p38 protein was then detected with anti-p38 or anti-p-38 antibodies by western blotting and quantified by p38/ACTB or p-p38/ACTB ratio. (C) p38 siRNA (10 nM) or sc siRNA (10 nM) were transfected into BV2 cells for 2 d. mRNA expression of TNF-α, iNOS, Nrf2, and COX-2 were quantified by qPCR. Data are expressed as the mean ± SEM (one-way ANOVA test, ***P < 0.001 vs sc siRNA + LPS).

Figure 3. Characterization of siRNA-encapsulated PLGA nanoparticles

(A–B) siRNA-encapsulated PLGA nanoparticles were dissolved in water and measured for size and zeta-potential using a Zetasizer. (C) Suspended nanoparticles were also assessed by TEM and SEM. Scale bar = 200 nm

Figure 4. Cytotoxicity and cellular uptake of PLGA nanoparticles

(A) BV2 cells were incubated with p38 siRNA-loaded PLGA nanoparticles (0–200 μg/mL) for 24 h and examined for cytotoxicity by MTT assay. Data are expressed as the mean ± SEM (one-way ANOVA test, n.s., not significant). (B) Coumarin 6-loaded PLGA nanoparticles were incubated for 3 h at 50 μg/mL in BV2 (microglia), HT22 (neurons), and U87MG (astrocytes) cells and examined under a fluorescent microscope to assess cellular uptake. (C) Two days after intrathecal injection of Coumarin 6 PLGA nanoparticles (C6 NPs, 200 μg in 20 μL), the L4-6 section of the spinal cord was excised and used for immunostaining with anti-NeuN (a neuronal marker), anti-GFAP (an astrocytic marker), or anti-OX42 (a microglial marker) antibodies to visualize the distribution of C6 NPs in the spinal cord. Scale bar = 50 μm (left), 20 μm (right). The number of C6 NPs-incorporated astrocytes, neurons and microglia was counted. Data are expressed as the mean ± SEM (one-way ANOVA test, ***P < 0.001 vs OX42).

Figure 5. Intrathecal injection of p38 siRNA-encapsulated PLGA nanoparticles attenuates mechanical hypersensitivity in SNL-induced rats

(A) Prior to SNL surgery, rats were subjected to a von Frey filament test with only those passing a pre-defined baseline (>10-g) selected for L5 SNL surgery. Following surgery, the von Frey test was repeated on days 3, 7, 10, and 14 post-injury. Data are presented as the mean ± SEM (Student t- test, ***P < 0.001 vs sham) (B) Spinal cord tissues (L5, POD7) were immunostained with anti-Iba1 antibodies (a microglia-specific marker). Magnified images represent sections taken from sites contralateral or ipsilateral to the dorsal horn of the spinal cord. Scale bar = 150 μm (upper), 100 μm (bottom) (C) Animals insensitive to the von Frey test (>10-g filament) were selected for SNL surgery. At day 7 post-SNL, sensitive rats (< 4-g filament) were divided into three groups (sc siRNA NPs, SB203580, and p38 siRNA NPs), with compound delivered directly to the lumber (L5–L6) intervertebral space by intrathecal injection. von Frey filament tests were performed on days 10, 12, and 14 post-surgery. Data are presented as the mean ± SEM (one-way ANOVA test; ***P < 0.001 vs sc siRNA NPs, ^###P < 0.001 vs SB203580; n.s. (not significant), p38 siRNA NPs vs. SB203580). 

Figure 6. p38 siRNA-encapsulated PLGA nanoparticles deactivate microglia and downregulate expression of genes downstream of p38 in the dorsal horns of the spinal cords in SNL-induced rats

sc siRNA- or p38 siRNA-loaded PLGA nanoparticles (NPs) were transferred into the lumber (L5–L6) intervertebral space of SNL-induced rats by intrathecal injection. (A) After 3 days, the L4–L5 section of the spinal cord was removed and used for immunostaining with anti-Iba1 antibodies. Magnified images (right) were taken from the ipsilateral dorsal horn of the spinal cord. Scale bar = 50 μm (left), 20 μm (right). (B) Total mRNA was isolated from the L4–L5 segment (0.7 cm) of the ipsilateral dorsal horn of the spinal cord at day 3 post-injection and utilized for cDNA synthesis. Expression of TNF-α, IL-1β, COX-2, and iNOS mRNA was then measured by qPCR. Data are presented as the mean ± SEM (one-way ANOVA test, *P < 0.5 vs sc siRNA NPs).

Figure 7. p38 siRNA-encapsulated PLGA nanoparticles decrease phosphorylation of p38 in microglia of the ipsilateral dorsal horn of the spinal cord in SNL-induced rats

(A) Frozen sections of spinal cords isolated from SNL-induced rats (POD7) were double-stained with anti-p-p38 and anti-OX-42 antibodies, anti-p-p38 and anti-NeuN antibodies, or anti-p-p38 and anti-GFAP antibodies. Scale bar = 50 μm (left), 20 μm (right). (B) The tissues used in Figure 6A were also co-immunostained with anti-p-p38 and anti-OX42 antibodies to identify the double-positive signals in microglia. Scale bar = 50 μm (left), 20 μm (right). The number of p-p38 or p-p38/OX42 positive cells in ipsilateral dorsal horns of spinal cords was counted. Data are expressed as the mean ± SEM (Student t- test, ***P < 0.001 vs sc NPs).