Investigation of adverse events following bacille Calmette-Guérin immunization using immunization safety surveillance system in Korea Centers for Disease Control and Prevention.

PURPOSE: The report of adverse events following immunization (AEFI) in Korea has continued since 1994, and the most frequently reported cases of AEFI of Korea Centers for Disease Control and Prevention (KCDC) is bacille Calmette-Guérin (BCG). Meanwhile, various inoculation methods and strains have been used in the past 6 years in Korea. Therefore, we investigated AEFI of BCG by strain types and inoculation methods using immunization safety surveillance of KCDC. MATERIALS AND METHODS: We reviewed BCG AEFIs registered in the KCDC from January 2013 to June 2018. RESULTS: There were 336 AEFI cases during the period, and average time interval from vaccination to symptom onset was within 2 months. AEFI proportion was 6.4 cases per 100,000 doses for BCG percutaneous Tokyo strain, 41.6 cases per 100,000 doses of BCG intradermal Danish strain, and 25.9 cases per 100,000 doses of BCG intradermal Tokyo strain. Intradermal type was more reported AEFI than percutaneous type in the same strain. The most common adverse events were local reaction like BCG lymphadenitis and severe adverse reactions such as osteomyelitis or disseminated BCG disease were 0.1 to 0.2 cases per 100,000 doses which are correlated with the range of World Health Organization published AEFI rates. CONCLUSION: The AEFI reporting rate does not equal the actual proportion of AEFI occurrence. Because AEFI monitoring is a passive surveillance system, various factors might influence the number of events reported. Nevertheless, it is important to analyze BCG AEFI by vaccine strains and inoculation method using surveillance data of KCDC.

Comparison of Marbling Fleck Characteristics and Objective Tenderness Parameters with Different Marbling Coarseness within Longissimus thoracis Muscle of High-marbled Hanwoo Steer.

It is important to understand how marbling traits and tenderness differ among beef steaks from the carcass grading site and other regions within the longissimus thoracis (LT) muscle, as these characteristics are closely associated with consumer acceptability and willingness to purchase. Thus, the aim of this study was to compare the marbling fleck traits and objective tenderness parameters in the groups classified by the coarseness index (CI) of marbling fleck (high and low groups) at the carcass grading site (13th thoracic vertebra; 13T) and three different locations (13T, 9T, and 6T) within the LT muscle from well-marbled (marbling score 7 to 9) Hanwoo steer. Image analysis showed that the longitudinal locations had a significant effect on marbling fleck traits. The total area of large marbling fleck divided by the total marbling area (coarseness) was higher at the central region (13T to 12T) compared to the front thoracic region (6T to 5T) in the high CI group (0.23 vs. 0.17, p<0.05), whereas no significant differences were observed in the total number of marbling fleck within the LT muscle in the high or low CI groups (p>0.05). Location effect on objective tenderness parameters within the LT muscle was somewhat limited, although the high CI group had a lower Warner-Bratzler shear force (WBS) value than did the low group (p<0.05). Taken together, the degree of coarseness of marbling fleck decreased from the carcass grading site to the front thoracic site, whereas the objective tenderness parameters, including WBS and hardness, of the grading site did not differ from the other regions within the LT muscle.

A phase I clinical study of autologous dendritic cell therapy in patients with relapsed or refractory multiple myeloma.

Cellular immunotherapy is emerging as a potential immunotherapeutic modality in multiple myeloma (MM). We have developed potent immunotherapeutic agent (VAX-DC/MM) generated by dendritic cells (DCs) loaded with autologous myeloma cells irradiated with ultraviolet B. In this study, we evaluated the safety and efficacy of VAX-DC/MM in patients with relapsed or refractory MM. This trial enrolled relapsed or refractory MM patients who had received both thalidomide- and bortezomib-based therapies. Patients received the intradermal VAX-DC/MM injection every week for 4 weeks. Patients were treated with 5 × 106 or 10 × 106 cells, with nine patients treated at a higher dose. The median time from diagnosis to VAX-DC/MM therapy was 56.6 months (range, 28.5-130.5). Patients had received a median of five prior treatments, and 75% had received autologous stem cell transplantation. VAX-DC therapy was well-tolerated, and the most frequent adverse events were local reactions at the injection site and infusion-related reactions. In seven of nine patients who received 10×106 cells, an immunological response (77.8%) was observed by interferon-gamma ELISPOT assay or a mixed lymphocyte reaction assay for T-cell proliferation. The clinical benefit rate was 66.7% including one (11.1%) with minor response and five (55.6%) with stable disease; three (33.3%) patients showed disease progression. In conclusion, VAX-DC/MM therapy was well-tolerated, and had disease-stabilizing activity in heavily pretreated MM cases. Further studies are needed to increase the efficacy of VAX-DC/MM in patients with MM.

Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in a Model of Murine Multiple Myeloma.

We investigated the efficacy of lenalidomide (LEN) in combination with dendritic cell (DC) vaccination in the MOPC-315 murine myeloma model. After tumor growth, LEN was injected intraperitoneally for 4 consecutive days in combination with DC vaccination. The combination of LEN and vaccination efficiently inhibited tumor growth compared with the single agents alone. A cytotoxic assay revealed that the anticancer effects of DC vaccination plus LEN involved not only generation of antigen-specific cytotoxic T lymphocytes but also NK cells. Vaccinated mice had reduced numbers of suppressor cells, including both myeloid-derived suppressor cells and regulatory T cells, in the spleen. The proportions of CD4+ and CD8+ T cells increased in the spleen, and a Th1 cytokine (interferon-γ) rather than a Th2 cytokine (interleukin-10) was synthesized in response to tumor antigens. LEN enhanced the innate immune response by modulating NK cell numbers and function. In addition, LEN reduced the production levels of angiogenesis-inducing factors in tumor-bearing mice. Together, these results suggest that a combination of LEN and DC vaccination may synergistically enhance anticancer immunity in the murine myeloma model, by inhibiting immunosuppressor cells and stimulating effector cells, as well as effectively polarizing the Th1/Th2 balance in favor of a Th1-specific immune response.

Dendritic Cell-Based Cancer Immunotherapy against Multiple Myeloma: From Bench to Clinic.

Although the introduction of stem cell transplantation and novel agents has improved survival, multiple myeloma (MM) is still difficult to cure. Alternative approaches are clearly needed to prolong the survival of patients with MM. Dendritic cell (DC) therapy is a very promising tool immunologically in MM. We developed a method to generate potent DCs with increased Th1 polarization and migration ability for inducing strong myeloma-specific cytotoxic T lymphocytes. In this review, we discuss how the efficacy of cancer immunotherapy using DCs can be improved in MM.

Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells.

Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCRαß(-)TCRγδ(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-γ. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells.

The relationship of spiritual well-being and involvement with depression and perceived stress in Korean nursing students.

UNLABELLED: This study was conducted to identify the relationship among spiritual well-being, depression and perceived stress. Participants were 518 nursing students located in K province, Korea. DESIGN: Exploratory design was used for this study. Data were collected and analyzed by t-test, ANOVA, Pearson correlation coefficients. The results were as follows; 1) Participants' mean scores were Spiritual Well-Being 76.03 (15.74), Religious Well-Being 32.8 (15.74), Existential Well-Being 43.23 (8.12), depression 9.10 (7.06), and level of stress 15.47 (5.49). 2) Spiritual Well-Being, Existential Well-Being, and Religious Well-Being were significantly different with the number of attendance to religious ceremony, the degree of subjective satisfaction regard to major and idea for future employment. 3) Negative correlations existed between Spiritual Well-Being and participants' perceived stress, and depression. 4) Particularly, Existential Well-Being has more inverse correlation with depression and stress than Religious Well-Being. This investigation highlighted Existential Well-Being as an important factor with lower levels of depression and perceived stress. According to these result, Spiritual Well-Being promotion program is needed to develop as a positive concept to adjust college life and spiritual well-being is required to take care of patients as potential power for nursing students in their future.

Generation of multiple peptide cocktail-pulsed dendritic cells as a cancer vaccine.

Cancer immunotherapy based on dendritic cell (DC) vaccination has promising alternatives for the treatment of cancer. A central tenet of DC-based cancer immunotherapy is the generation of antigen-specific cytotoxic T lymphocyte (CTL) response. Tumor-associated antigens (TAA) and DC play pivotal roles in this process. DCs are well known to be the most potent antigen-presenting cells and have the most powerful antigen-presenting capacity. DCs pulsed with various TAA have been shown to be effective in producing specific antitumor effects both in vitro and in vivo. Several types of tumor antigens have been applied in cancer treatment including tumor RNA, lysates, apoptotic bodies, heat shock protein, peptides from TAA, and allogeneic tumor cells. Among them, the use of immunogenic HLA-A*0201-specific epitopes from multiple TAA enhances induction of antigen-specific CTL and associated therapeutic efficacy in HLA-A*0201(+) cancer patients. The current chapter provides a detailed protocol of generating multiple peptide cocktail-pulsed DC to elicit CTL with a broad spectrum of immune responses against the related tumor antigens.

Dendritic cells loaded with myeloma cells pretreated with a combination of JSI-124 and bortezomib generate potent myeloma-specific cytotoxic T lymphocytes in vitro.

Signal transducer and activator of transcription 3 (STAT3) is highly activated in multiple myeloma. Activated STAT3 promotes survival and proliferation of cancer cells, suppresses Th1 immune response, and induces dysfunction of immune cells. We investigated whether pretreating myeloma cells with a phosphor (p)-STAT3 inhibitor (JSI-124) and/or bortezomib before loading into dendritic cells (DCs) can affect DC function. The combination treatment with JSI-124 and bortezomib resulted in the highest expression of heat shock protein (HSP) 90 and the lowest expression of p-STAT3 in dying myeloma cells. DCs loaded with dying myeloma cells treated by JSI-124 and bortezomib produced the least amount of p-STAT3 compared to other treatments. The DCs were recovered from abnormal cytokine secretions of interleukin (IL)-10, IL-6, and IL-23 without any effect on production of IL-12p70. DCs loaded with JSI-124 and bortezomib treated, dying myeloma cells most potently generated myeloma-specific cytotoxic T lymphocytes (CTLs). The data suggest that pretreatment of myeloma cells with JSI-124 and bortezomib can recover DC function through the up-regulation of HSP90 and the down-regulation of p-STAT3 and inhibitory cytokines, and that these DCs can potently generate myeloma-specific CTLs.

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells.

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαß and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3(+)CD8(+) TCRαß(-)TCRγδ(-)CD4(-)CD21(-)CD11c(+/-)CD11d(+/-)CD44(+). The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy.

Structures and properties of poly(3-alkylthiophene) thin-films fabricated though vapor-phase polymerization.

Organic semiconducting polymer thin-films of 3-hexylthiophene, 3-octylthiophene, 3-decylthiophene, containing highly oriented crystal were fabricated by gas-phase polymerization using the CVD technique. These poly(3-alkylthiophene) films had a crystallinity up to 80%, and possessed a Hall mobility up to 10 cm2/Vs. The degree of crystalinity and the mobility values increased as the alkyl chain length increased. The crystal structure of the polymers was composed of stacked layers constructed by a side-by-side arrangement of alkyl chains and in-plane pi-pi stacking. These thin films are capable of being applied to organic electronics as the active materials used in thin-film transistors and organic photovoltaic cells.

Cellular immunotherapy using dendritic cells against multiple myeloma.

Cellular therapy with dendritic cells (DCs) is emerging as a useful immunotherapeutic tool to treat multiple myeloma (MM). DC-based idiotype vaccination was recently suggested to induce idiotype-specific immune responses in MM patients. However, the clinical results so far have been largely disappointing, and the clinical effectiveness of such vaccinations in MM still needs to be demonstrated. DC-based therapies against MM may need to be boosted with other sources of tumor-associated antigens, and potent DCs should be recruited to increase the effectiveness of treatment. DCs with both high migratory capacity and high cytokine production are very important for effective DC-based cancer vaccination in order to induce high numbers of Th1-type CD4(+) T cells and CD8(+) cytotoxic T lymphocytes. The tumor microenvironment is also important in the regulation of tumor cell growth, proliferation, and the development of therapeutic resistance after treatment. In this review, we discuss how the efficacy of DC vaccination in MM can be improved. In addition, novel treatment strategies that target not only myeloma cells but also the tumor microenvironment are urgently needed to improve treatment outcomes.

Identification of novel HLA-A*0201-restricted epitopes from anterior gradient-2 as a tumor-associated antigen against colorectal cancer.

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration and cellular transformation and its enhanced expression is almost completely restricted to malignant tissues, thus making AGR2 an interesting target for the development of immunotherapeutic strategies. We investigated whether the AGR2 molecule comprises human leukocyte antigen (HLA)-A*0201-binding epitopes recognized by human cytotoxic T lymphocytes (CTLs), which could be targeted in dendritic cell (DC)-based cancer immunotherapy against colorectal cancer (CRC). We reviewed the sequence of AGR2 for peptides that could potentially bind to HLA-A*0201 with the aid of a computer-based program. Five candidate peptides with different binding scores were synthesized and tested. These peptides were then assessed for their immunogenicity to elicit specific immune responses mediated by CTLs in vitro by means of enzyme-linked immunospot assays and CTL assays. AGR2 was highly expressed in several CRC cell lines, including DK01, DLD1, KM12C, HCT-8 and HT-29. DCs pulsed with AGR2-P2 (aa 11-19; LLVALSYTL) or AGR2-P4 (aa 127-135; RIMFVDPSL) generated potent CTLs that could lyse T2 cells pulsed with AGR2-P2 or AGR2-P4 and HLA-A0201(+) AGR2-positive CRC cell lines in a strong dose-dependent and HLA-A*0201-restricted manner. In conclusion, these novel epitopes derived from AGR2 protein may be attractive candidates for DC-based immunotherapy for CRC.

Optimal culture conditions for the generation of natural killer cell-induced dendritic cells for cancer immunotherapy.

Dendritic cell (DC)-based vaccines continue to be considered an attractive tool for cancer immunotherapy. DCs require an additional signal from the environment or other immune cells to polarize the development of immune responses toward T helper 1 (Th1) or Th2 responses. DCs play a role in natural killer (NK) cell activation, and NK cells are also able to activate and induce the maturation of DCs. We investigated the types of NK cells that can induce the maturation and enhanced function of DCs and the conditions under which these interactions occur. DCs that were activated by resting NK cells in the presence of inflammatory cytokines exhibited increased expression of several costimulatory molecules and an enhanced ability to produce IL-12p70. NK cell-stimulated DCs potently induced Th1 polarization and exhibited the ability to generate tumor antigen-specific cytotoxic T lymphocyte responses. Our data demonstrate that functional DCs can be generated by coculturing immature DCs with freshly isolated resting NK cells in the presence of Toll-like receptor agonists and proinflammatory cytokines and that the resulting DCs effectively present antigens to induce tumor-specific T-cell responses, which suggests that these cells may be useful for cancer immunotherapy.

Induction of myeloma-specific cytotoxic T lymphocytes responses by natural killer cells stimulated-dendritic cells in patients with multiple myeloma.

The interaction between dendritic cells (DCs) and natural killer (NK) cells plays a key role in inducing DC maturation for subsequent T-cell priming. We investigated to generate potent DCs by stimulated with NK cells to induce myeloma-specific cytotoxic T lymphocytes (CTLs). NK cells-stimulated-DCs exhibited high expression of costimulatory molecules and high production of IL-12p70. These DCs induce high potency of Th1 polarization and exhibit a high ability to generate myeloma-specific CTLs responses. These results suggest that functionally potent DCs can be generated by stimulation with NK cells and may provide an effective source of DC-based immunotherapy in multiple myeloma.

Successful cross-presentation of allogeneic myeloma cells by autologous alpha-type 1-polarized dendritic cells as an effective tumor antigen in myeloma patients with matched monoclonal immunoglobulins.

For wide application of a dendritic cell (DC) vaccination in myeloma patients, easily available tumor antigens should be developed. We investigated the feasibility of cellular immunotherapy using autologous alpha-type 1-polarized dendritic cells (αDC1s) loaded with apoptotic allogeneic myeloma cells, which could generate myeloma-specific cytotoxic T lymphocytes (CTLs) against autologous myeloma cells in myeloma patients. Monocyte-derived DCs were matured by adding the αDC1-polarizing cocktail (TNFα/IL-1ß/IFN-α/IFN-γ/poly-I:C) and loaded with apoptotic allogeneic CD138(+) myeloma cells from other patients with matched monoclonal immunoglobulins as a tumor antigen. There were no differences in the phenotypic expression between αDC1s loaded with apoptotic autologous and allogeneic myeloma cells. Autologous αDC1s effectively took up apoptotic allogeneic myeloma cells from other patients with matched subtype. Myeloma-specific CTLs against autologous target cells were successfully induced by αDC1s loaded with allogeneic tumor antigen. The cross-presentation of apoptotic allogeneic myeloma cells to αDC1s could generate CTL responses between myeloma patients with individual matched monoclonal immunoglobulins. There was no difference in CTL responses between αDC1s loaded with autologous tumor antigen and allogeneic tumor antigen against targeting patient's myeloma cells. Our data indicate that autologous DCs loaded with allogeneic myeloma cells with matched immunoglobulin can generate potent myeloma-specific CTL responses against autologous myeloma cells and can be a highly feasible and effective method for cellular immunotherapy in myeloma patients.

Alpha-Type 1 Polarized Dendritic Cells Loaded with Apoptotic Allogeneic Breast Cancer Cells Can Induce Potent Cytotoxic T Lymphocytes against Breast Cancer.

PURPOSE: Various tumor antigens can be loaded onto dendritic cells (DCs) to induce a potent cytotoxic T lymphocyte (CTL) response in DC-based immunotherapy against breast cancer. However, in the clinical setting, obtaining a sufficient number of autologous tumor cells as a source of tumor antigens is a laborious process. We therefore investigated the feasibility of immunotherapy using breast-cancer-specific CTLs generated in vitro by use of alpha-type 1 polarized DCs (α DC1s) loaded with ultraviolet B-irradiated cells of the breast cancer cell line MCF-7. MATERIALS AND METHODS: αDC1s were induced by loading allogeneic tumor antigen generated from the MCF-7 UVB-irradiated breast cancer cell line. Antigen-pulsed αDC1s were evaluated by morphological and functional assays, and the breast-cancer-specific CTL response was analyzed by cytotoxic assay. RESULTS: The αDC1s significantly increased the expression of several molecules related to DC maturation without differences according to whether the αDC1s were loaded with tumor antigens. The αDC1s showed a high production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens. Breast-cancer-specific CTLs against autologous breast cancer cells were successfully induced by αDC1s loaded with apoptotic MCF-7 cells. CONCLUSION: Autologous DCs loaded with an allogeneic breast cancer cell line can generate potent breast-cancer-specific CTL responses. This may be a practical method for cellular immunotherapy in patients with breast cancer.

Type I and II interferons enhance dendritic cell maturation and migration capacity by regulating CD38 and CD74 that have synergistic effects with TLR agonists.

The major limitation for the maturation of dendritic cells (DCs) using Toll-like receptor (TLR) agonists is their decreased ability to migrate into lymph nodes compared with conventional DCs. CD38 can be used as a multifunctional marker to modulate migration, survival and Th1 responses of DCs. CD74 has been shown to negatively regulate DC migration. The goal of this study was to investigate the combinations of TLR agonists and interferons (IFNs) that most effectively regulate CD38 and CD74 expression on DCs. Synergistic TLR agonist stimulation in combination with IFN-α and IFN-γ was the best method for regulating CD38 and CD74 expression and inducing the highest secretion of IL-12p70. An in vitro migration assay showed that DCs treated with this combination had significantly enhanced migratory ability, similar to that observed in cells expressing CD38, CD74 and CCR7. The results of this study suggest that an alternative maturation protocol in which two TLR ligands are combined with type I and II IFNs generates potent DCs that have both a high migratory capacity and high IL-12p70 production.

Alpha-type 1-polarized dendritic cells loaded with apoptotic allogeneic myeloma cell line induce strong CTL responses against autologous myeloma cells.

To induce a potent cytotoxic T lymphocyte (CTL) response, various tumor antigens should be loaded onto dendritic cells (DCs). In multiple myeloma (MM), it is difficult to obtain a sufficient number of autologous tumor cells as a source of tumor antigens in the clinical setting. We investigated the feasibility of immunotherapy in patients with MM, using myeloma-specific CTLs generated in vitro by alpha-type 1-polarized DCs (alphaDC1s) loaded with the ultraviolet B-irradiated allogeneic myeloma cell line, ARH77. alphaDC1s significantly increased the expression of several costimulatory molecules without differences in loading with tumor antigens. alphaDC1s showed a high production of interleukin-12 during maturation and after subsequent stimulation with CD40L but were not significantly affected by loading tumor antigens. Myeloma-specific CTLs against autologous myeloma cells from MM patients were induced by alphaDC1s pulsed with apoptotic ARH77 cells. Our data indicate that autologous DCs loaded with an allogeneic myeloma cell line can generate potent myeloma-specific CTL responses against autologous myeloma cells and might provide a practical method for cellular immunotherapy in patients with MM.

Efficient revascularization by VEGF administration via heparin-functionalized nanoparticle-fibrin complex.

We investigated the angiogenic bioactivity and therapeutic angiogenic effect of vascular endothelial growth factor (VEGF) administration by the heparin-functionalized nanoparticle-fibrin gel complex. The markedly increased bioactivity was observed by the VEGF-loaded nanoparticle-fibrin gel complex, compared to the VEGF-loaded fibrin gel, the nanoparticle-fibrin gel complex without VEGF, or fibrin gel (control) in terms of the capillary density in a mouse subcutaneous implantation model. Furthermore, the VEGF-loaded nanoparticle-fibrin gel complex significantly enhanced the therapeutic angiogenic effect in a rabbit ischemic hind limb model: the noticeable increase in the recovered calf blood pressure, the angiographic score, and the density of collaterals, as well as the stable maintenance of the organized collaterals, compared to the VEGF-loaded fibrin gel. These results show the enhanced angiogenic potential of VEGF administration by the proposed heparin-functionalized nanoparticle-fibrin gel complex.