Biosafety of Non-Surface Modified Carbon Nanocapsules as a Potential Alternative to Carbon Nanotubes for Drug Delivery Purposes

BACKGROUND: Carbon nanotubes (CNTs) have found wide success in circuitry, photovoltaics, and other applications. In contrast, several hurdles exist in using CNTs towards applications in drug delivery. Raw, non-modified CNTs are widely known for their toxicity. As such, many have attempted to reduce CNT toxicity for intravenous drug delivery purposes by post-process surface modification. Alternatively, a novel sphere-like carbon nanocapsule (CNC) developed by the arc-discharge method holds similar electric and thermal conductivities, as well as high strength. This study investigated the systemic toxicity and biocompatibility of different non-surface modified carbon nanomaterials in mice, including multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), carbon nanocapsules (CNCs), and C ₆₀ fullerene (C ₆₀). The retention of the nanomaterials and systemic effects after intravenous injections were studied. METHODOLOGY AND PRINCIPAL FINDINGS: MWCNTs, SWCNTs, CNCs, and C ₆₀ were injected intravenously into FVB mice and then sacrificed for tissue section examination. Inflammatory cytokine levels were evaluated with ELISA. Mice receiving injection of MWCNTs or SWCNTs at 50 µg/g b.w. died while C ₆₀ injected group survived at a 50% rate. Surprisingly, mortality rate of mice injected with CNCs was only at 10%. Tissue sections revealed that most carbon nanomaterials retained in the lung. Furthermore, serum and lung-tissue cytokine levels did not reveal any inflammatory response compared to those in mice receiving normal saline injection. CONCLUSION: Carbon nanocapsules are more biocompatible than other carbon nanomaterials and are more suitable for intravenous drug delivery. These results indicate potential biomedical use of non-surface modified carbon allotrope. Additionally, functionalization of the carbon nanocapsules could further enhance dispersion and biocompatibility for intravenous injection

Carbon nanomaterial retention in the lungs.

<p>(<b>A</b>) Lung tissue sections of mice 7 days after intravenous injection with carbon nanomaterials at 25 μg/g b.w. (<b>B</b>) Automatic carbon nanomaterial retention quantification in the lungs. High-magnification images (red-bordered images) show large carbon nanotube aggregates blocking the blood vessels of the lungs (arrows). SWCNTs and MWCNTs were retained in the lungs at much higher rates compared to CNCs or C₆₀. Tissue sections were stained with hematoxylin. Scale bar = 100 μm. ***<i>P</i><0.0001 compared to CNCs and C₆₀, n = 4 in all groups. NS, normal saline; PVA, polyvinyl alcohol; CNCs, carbon nanocapsules; C₆₀, C₆₀ fullerene; MWCNTs, multi-walled carbon nanotubes; SWCNTs, single-walled carbon nanotubes.</p

Systemic inflammatory cytokine level in mice.

<p>Serum and lung tissue IL-1β and IL-6 levels 6 hours post-injection with carbon nanomaterials. There is no significant difference between all groups (except LPS). n = 5, <i>P</i> = 0.0029 (serum, IL-1β); n = 5, <i>P</i> = 0.0001 (serum, IL-6); n = 4. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 significantly different compared with the LPS group. NS, normal saline; PVA, polyvinyl alcohol; CNCs, carbon nanocapsules; C₆₀, C₆₀ fullerene; M, multi-walled carbon nanotubes; S, single-walled carbon nanotubes.</p

Mouse survival curves after carbon nanomaterial injection.

<p>(<b>A</b>) TEM analysis of carbon nanocapsules (CNCs), C₆₀ fullerene (C₆₀), multi-walled carbon nanotubes (MWCNTs), and single-walled carbon nanotubes (SWCNTs) dispersed in 1% polyvinyl alcohol (PVA). SWCNTs formed large networks, and MWCNTs aggregated compactly. CNCs were well dispersed in PVA, while C₆₀ aggregated to size as large as CNCs. The scale bar is 100<b> </b>nm. (<b>B</b>) Cumulative deaths of mice intravenously injected with different doses of carbon nanomaterials. SWCNTs and MWCNTs had the highest toxicity, which was dose dependent, decreasing as the dose of the carbon nanomaterials decreased. No mortality was observed among the CNC-treated mice at 25 μg/g b.w. n = 12 for CNC, and C₆₀ injected mice. n = 11 for NS, PVA, MWCNT, and SWCNT injected mice. Red square, 50 μg/g; black dot, 25 μg/g; black cross, 12.5 μg/g.</p

Lung tissue inflammatory cytokine level in mice.

<p>Lung tissue IL-1β and IL-6 levels 6 hours post-injection with carbon nanomaterials. There is no significant difference between all groups (except LPS). Lung-tissue cytokine levels were normalized to the total protein level determined using a BCA kit (Pierce, USA). n = 5, ***<i>P</i> = 0.0001 (lung tissue, IL-1β); n = 4, <i>P</i> = 0.0001 (lung tissue, IL-6), compared to the LPS group. NS, normal saline; PVA, polyvinyl alcohol; CNCs, carbon nanocapsules; C₆₀, C₆₀ fullerene; M, multi-walled carbon nanotubes; S, single-walled carbon nanotubes.</p

Carbon nanomaterial retention in vital organs.

<p>Liver, spleen, and kidney tissue sections of mice 7 days after intravenous injection with carbon nanomaterials at 25 μg/g b.w. Carbon nanomaterials are indicated by arrows. Tissue sections were stained with hematoxylin. The scale bar is 50 μm. PVA, polyvinyl alcohol; CNCs, carbon nanocapsules; C₆₀, C₆₀ fullerene; MWCNTs, multi-walled carbon nanotubes; SWCNTs, single-walled carbon nanotubes.</p

Lung tissues and lung tissue sections after carbon nanomaterial injection.

<p>(<b>A</b>) Excised lungs 10 min after mice were injected with 50 μg/g b.w. of different carbon nanomaterials. (<b>B</b>) Lung tissue sections 10 min after mice were injected with 50 μg/g b.w. of different carbon nanomaterials. Mice receiving C₆₀ fullerene (C₆₀), multi-walled carbon nanotubes (MWCNTs), and single-walled carbon nanotubes (SWCNTs) died within 10 minutes, and only mice in the carbon nanocapsule (CNC), normal saline, and polyvinyl alcohol (PVA) groups had to be sacrificed. Tissue sections were stained with hematoxylin. The scale bar is 400 μm.</p

Instructive nanofiber scaffolds with VEGF create a microenvironment for arteriogenesis and cardiac repair.

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation

Instructive nanofiber scaffolds with VEGF create a microenvironment for arteriogenesis and cardiac repair.

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation

The Time Window for Therapy with Peptide Nanofibers Combined with Autologous Bone Marrow Cells in Pigs after Acute Myocardial Infarction

<div><p>Background</p><p>We previously showed that injection of peptide nanofibers (NF) combined with autologous bone marrow mononuclear cells (MNC) immediately after coronary artery ligation improves cardiac performance in pigs. To evaluate the clinical feasibility, this study was performed to determine the therapeutic time window for NF/MNC therapy in acute myocardial infarction (MI).</p><p>Methods and Results</p><p>A total of 45 adult minipigs were randomly grouped into 7 groups: sham or MI plus treatment with NS (normal saline), or NF or MNC alone at 1 day (1D) post-MI, or NF/MNC at 1, 4, or 7 days post-MI (N≥6). Cardiac function was assessed by echocardiography and ventricular catheterization. Compared with the NS control, pigs treated with NF/MNC at 1 day post-MI (NF/MC-1D) had the greatest improvement in left ventricle ejection fraction (LVEF; 55.1±1.6%; P<0.01 vs. NS) 2 months after MI. In contrast, pigs treated with either NF/MNC-4D or NF/MNC-7D showed 48.9±0.8% (P<0.05 vs. NS) and 43.5±2.3% (n.s. vs. NS) improvements, respectively. The +dP/dt and -dP/dt, infarct size and interstitial collagen content were also improved in the NF/MNC-1D and -4D groups but not in the -7D group. Mechanistically, MNC quality and the states of systemic inflammation and damaged heart tissue influence the therapeutic efficiency of NF/MNC therapy, as revealed by another independent study using 16 pigs.</p><p>Conclusions</p><p>Injection of NF/MNC at 1 or 4 days, but not at 7 days post-MI, improves cardiac performance and prevents ventricular remodeling, confirming the importance of early intervention when using this therapy for acute MI.</p></div