Treatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury

doi:10.1016/j.nano.2020.102247

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 29, October 2020, 102247

https://doi.org/10.1016/j.nano.2020.102247 Get rights and content

Highlights

•
Repeated TBI (rTBI) causes neuroinflammation in the brain.
•
PAMAM nanodendriplexes effectively deliver plasmid DNA encoding shRNAs of CCL20 and/or CCR6 to the brain and spleen.
•
Treatment with shCCL20-CCR6 nanodendriplexes reduces the rTBI-induced neuroinflammation.
•
A combination therapy involving shCCL20-CCR6 nanodendriplexes followed by human mesenchymal stem cells (hMSCs) increased BDNF and better reduced TBI burden vs single treatments.
•
Thus, a prior reduction of inflammatory microenvironment improves the efficacy of hMSC transplantation in rTBI mice.

Abstract

Traumatic brain injury (TBI) is a devastating neurological disorder, although the underlying pathophysiology is poorly understood. TBI causes blood–brain barrier (BBB) disruption, immune cell trafficking, neuroinflammation and neurodegeneration. CCL20 is an important chemokine mediating neuroinflammation. Human mesenchymal stem cell (hMSC) therapy is a promising regenerative approach but the inflammatory microenvironment in the brain tends to decrease the efficacy of the hMSC transplantation. Reducing the inflammation prior to hMSC therapy improves the outcome. We developed a combined nano-cell therapy by using dendrimers complexed with plasmids (dendriplexes) targeting CCL20 and its sole receptor CCR6 to reduce inflammation followed by hMSC transplantation. Treatment of TBI mice with shRNA conjugated dendriplexes followed by hMSC administration downregulated the inflammatory markers and significantly increased brain-derived neurotrophic factor (BDNF) expression in the cerebral cortex indicating future possible neurogenesis and improved behavioral deficits. Taken together, this nano-cell therapy ameliorates neuroinflammation and promotes brain tissue repair after TBI.

Graphical Abstract

Herein we describe the development of a nanodendriplex comprising PAMAM dendrimers with plasmids as the payload encoding small-interfering RNAs of the chemokine CCL20 and its sole receptor CCR6. The intranasal and intravenous administration of these nanodendriplexes followed by human mesenchymal stem cell (hMSC) treatment significantly improved the pathological and behavioral outcomes in repeated traumatic brain injury (rTBI) mice. We provide evidence that while nanodendriplexes down-regulated CCL20-CCR6 expression and pro-inflammatory cytokine, IL-6, the combination of nanodendriplexes and hMSC significantly increased BDNF expression indicating possible neurogenesis.

Section snippets

Construction of CCL20/CCR6 shRNA expression vector

Wizard 3.1 software from Invivogen (San Diego, USA) was used for designing the shRNA target sequence for knocking down CCL20 and CCR6. The mammalian multiple miR30-shRNA knockdown vectors for downregulating CCL20 or CCR6 were purchased from Vector Builder (VectorBuilder lnc, Chicago, USA). Each vector is encoded with 4 inserts of shRNA (CCL20 or CCR6) sequence with (pRP[miR30-shRNA]-Neo-CMV > TurboRFP) CMV promoter, turbo RFP reporter and ampicillin as selectable marker gene. Similarly, a

Preparation and characterization of dendriplex nanoparticles

PAMAM G4 dendrimer was first labeled with Cy-7 utilizing the amine groups on the dendrimer surface and then complexed with shRNA encoding plasmid DNA (Figure 1, A). The successful DNA/dendrimer complexation was confirmed using agarose gel electrophoresis. The average size and ζ-potential of the dendrimer and dendriplex were measured in ultrapure water using zetasizer. Figure 1, B shows that the initial average hydrodynamic diameter of PAMAM dendrimer was 5 nm (red line) and after complexation

Discussion

rTBI induces significant neurodegeneration and tissue loss in the cerebral cortex 7 days post injury in mice. Activation of microglia, astrocytes and cytokine production indicates an active inflammatory milieu in the brain post rTBI. To recover from the damage, it is important to prevent the secondary spread of damage as well as stimulate neurogenesis to enhance the recovery process. Treating TBI conditions with drugs has not been successful so far. Drugs like erythropoietin¹³^,¹⁴ and

Acknowledgment

This work is supported by a Veterans Affairs Merit Review grant (BX002668) to Subhra Mohapatra, and Research Career Scientist Awards to Dr. Subhra Mohapatra (IK6BX004212) and Dr. Shyam Mohapatra (IK6 BX003778). Though this report is based upon work supported, in part, by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, the contents of this report do not represent the views of the Department of Veterans Affairs or the United States

Authors' contributions

SM, SSM and AW designed the experiments. EM and KM designed and produced the nanoparticles. KM and MD conducted experimental studies and wrote the manuscript. PC, DP, AK helped in tissue processing, immunostaining and qPCR, SM, AW, DG and SSM reviewed the manuscript.

References (64)

B.E. Wojcik
Traumatic brain injury hospitalizations of U.S. army soldiers deployed to Afghanistan and Iraq
Am J Prev Med
(2010)
A.C. McKee et al.
Military-related traumatic brain injury and neurodegeneration
Alzheimers Dement
(2014)
A. Nichol
Erythropoietin in traumatic brain injury (EPO-TBI): a double-blind randomised controlled trial
Lancet
(2015)
P. Mastorakos
Biodegradable brain-penetrating DNA nanocomplexes and their use to treat malignant brain tumors
J Control Release
(2017)
E.D. Hall et al.
Antioxidant therapies for traumatic brain injury
Neurotherapeutics
(2010)
S.F. Chen
Lovastatin improves histological and functional outcomes and reduces inflammation after experimental traumatic brain injury
Life Sci
(2007)
L.M. Buja et al.
Immunologic and inflammatory reactions to exogenous stem cells implications for experimental studies and clinical trials for myocardial repair
J Am Coll Cardiol
(2010)
E. Schutyser et al.
The CC chemokine CCL20 and its receptor CCR6
Cytokine Growth Factor Rev
(2003)
W. Qi
The roles of Kruppel-like factor 6 and peroxisome proliferator-activated receptor-gamma in the regulation of macrophage inflammatory protein-3alpha at early onset of diabetes
Int J Biochem Cell Biol
(2011)
D.W. Sah
Therapeutic potential of RNA interference for neurological disorders
Life Sci
(2006)

I.D. Kim

Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain

J Control Release

(2010)

K. Karolczak

Poly(amido)amine dendrimers generation 4.0 (PAMAM G4) reduce blood hyperglycaemia and restore impaired blood-brain barrier permeability in streptozotocin diabetes in rats

Int J Pharm

(2012)

T.T. Win-Shwe

Effects of PAMAM dendrimers in the mouse brain after a single intranasal instillation

Toxicol Lett

(2014)

M.V. Sofroniew

Molecular dissection of reactive astrogliosis and glial scar formation

Trends Neurosci

(2009)

A. Hasan

Mesenchymal stem cells in the treatment of traumatic brain injury

Front Neurol

(2017)

G. Laviola

Risk-taking behavior in adolescent mice: psychobiological determinants and early epigenetic influence

Neurosci Biobehav Rev

(2003)

Peterson, A.B., Xu Likang, Daugherty, Jill, Breiding, Matthew J., Surveillance report of traumatic brain injury-related...

K. Draper et al.

Cognitive functioning ten years following traumatic brain injury and rehabilitation

Neuropsychology

(2008)

J. Ponsford et al.

Functional outcome 10 years after traumatic brain injury: its relationship with demographic, injury severity, and cognitive and emotional status

J Int Neuropsychol Soc

(2008)

L.K. Lindquist et al.

Traumatic brain injury in Iraq and Afghanistan veterans: new results from a national random sample study

J Neuropsychiatry Clin Neurosci

(2017)

C.J. Bryan et al.

Repetitive traumatic brain injury, psychological symptoms, and suicide risk in a clinical sample of deployed military personnel

JAMA Psychiat

(2013)

J. Popovitz et al.

Long-term effects of traumatic brain injury on anxiety-like behaviors in mice: behavioral and neural correlates

Front Behav Neurosci

(2019)

C.W. Hoge

Mild traumatic brain injury in U.S. soldiers returning from Iraq

N Engl J Med

(2008)

H. Terrio

Traumatic brain injury screening: preliminary findings in a US army brigade combat team

J Head Trauma Rehabil

(2009)

M. Carbonara

Neuroprotection in traumatic brain injury: mesenchymal stromal cells can potentially overcome some limitations of previous clinical trials

Front Neurol

(2018)

C.S. Cox

J. Juranek, and S. Bedi, Clinical trials in traumatic brain injury: cellular therapy and outcome measures

Transfusion

(2019)

C.S. Robertson

Effect of erythropoietin and transfusion threshold on neurological recovery after traumatic brain injury: a randomized clinical trial

JAMA

(2014)

B.E. Skolnick

A clinical trial of progesterone for severe traumatic brain injury

N Engl J Med

(2014)

L.T. Galindo

Mesenchymal stem cell therapy modulates the inflammatory response in experimental traumatic brain injury

Neurol Res Int

(2011)

A.M. Parr et al.

Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury

Bone Marrow Transplant

(2007)

E. Redondo-Castro

Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

Stem Cell Res Ther

(2017)

D. Garcia-Olmo

A phase I clinical trial of the treatment of Crohn's fistula by adipose mesenchymal stem cell transplantation

Dis Colon Rectum

(2005)

Cited by (15)

Microencapsulated stem cells reduce cartilage damage in a material dependent manner following minimally invasive intra-articular injection in an OA rat model
2023, Materials Today Bio
Osteoarthritis (OA) is a degenerative disease of the joints for which no curative treatment exists. Intra-articular injection of stem cells is explored as a regenerative approach, but rapid clearance of cells from the injection site limits the therapeutic outcome. Microencapsulation of mesenchymal stem cells (MSCs) can extend the retention time of MSCs, but the outcomes of the few studies currently performed are conflicting. We hypothesize that the composition of the micromaterial's shell plays a deciding factor in the treatment outcome of intra-articular MSC injection. To this end, we microencapsulate MSCs using droplet microfluidic generators in flow-focus mode using various polymers and polymer concentrations. We demonstrate that polymer composition and concentration potently alter the metabolic activity as well as the secretome of MSCs. Moreover, while microencapsulation consistently prolongs the retention time of MSC injected in rat joints, distinct biodistribution within the joint is demonstrated for the various microgel formulations. Furthermore, intra-articular injections of pristine and microencapsulated MSC in OA rat joints show a strong material-dependent effect on the reduction of cartilage degradation and matrix loss. Collectively, this study highlights that micromaterial composition and concentration are key deciding factors for the therapeutic outcome of intra-articular injections of microencapsulated stem cells to treat degenerative joint diseases.
A mouse model of repeated traumatic brain injury-induced hearing impairment: Early cochlear neurodegeneration in the absence of hair cell loss
2023, Hearing Research
Traumatic Brain Injury (TBI) is a major cause of death and disability worldwide. Mounting evidence suggests that even mild TBI injuries, which comprise >75% of all TBIs, can cause chronic post-concussive neurological symptoms, especially when experienced repetitively (rTBI). The most common post-concussive symptoms include auditory dysfunction in the form of hearing loss, tinnitus, or impaired auditory processing, which can occur even in the absence of direct damage to the auditory system at the time of injury. The mechanism by which indirect damage causes loss of auditory function is poorly understood, and treatment is currently limited to symptom management rather than preventative care. We reasoned that secondary injury mechanisms, such as inflammation, may lead to damage of the inner ear and parts of the brain used for hearing after rTBI. Herein, we established a model of indirect damage to the auditory system induced by rTBI and characterized the pathology of hearing loss.
We established a mouse model of rTBI in order to determine a timeline of auditory pathology following multiple mild injuries. Mice were subject to controlled cortical impact at the skull midline once every 48 h, for a total of 5 hits. Auditory function was assessed via the auditory brainstem response (ABR) at various timepoints post injury. Brain and cochleae were collected to establish a timeline of cellular pathology.
We observed increased ABR thresholds and decreased (ABR) P1 amplitudes in rTBI vs sham animals at 14 days post-impact (dpi). This effect persisted for up to 60 days (dpi). Auditory temporal processing was impaired beginning at 30 dpi. Spiral ganglion degeneration was evident at 14 dpi. No loss of hair cells was detected at this time, suggesting that neuronal loss is one of the earliest notable events in hearing loss caused by this type of rTBI.
We conclude that rTBI results in chronic auditory dysfunction via damage to the spiral ganglion which occurs in the absence of any reduction in hair cell number. This suggests early neuronal damage that may be caused by systemic mechanisms similar to those leading to the spread of neuronal death in the brain following TBI. This TBI-hearing loss model provides an important first step towards identifying therapeutic targets to attenuate damage to the auditory system following head injury.
Multispectral optoacoustic tomography (MSOT): Monitoring neurovascular changes in a mouse repetitive traumatic brain injury model
2023, Journal of Neuroscience Methods
Evidence suggests that mild TBI injuries, which comprise > 75% of all TBIs, can cause chronic post-concussive symptoms, especially when experienced repetitively (rTBI). rTBI is a major cause of cognitive deficit in athletes and military personnel and is associated with neurovascular changes. Current methods to monitor neurovascular changes in detail are prohibitively expensive and invasive for patients with mild injuries.
We evaluated the potential of multispectral optoacoustic tomography (MSOT) to monitor neurovascular changes and assess therapeutic strategies in a mouse model of rTBI. Mice were subjected to rTBI or sham via controlled cortical impact and administered pioglitazone (PG) or vehicle. Oxygenated and deoxygenated hemoglobin were monitored using MSOT. Indocyanine green clearance was imaged via MSOT to evaluate blood-brain-barrier (BBB) integrity.
Mice subjected to rTBI show a transient increase in oxygenated/total hemoglobin ratio which can be mitigated by PG administration. rTBI mice also show BBB disruption shortly after injury and reduction of oxygenated/total hemoglobin in the chronic stage, neither of which were affected by PG intervention.
MSOT imaging has the potential as a noninvasive in vivo imaging method to monitor neurovascular changes and assess therapeutics in mouse models of rTBI. In comparison to standard methods of tracking inflammation and BBB disruption, MSOT can be used multiple times throughout the course of injury without the need for surgery. Thus, MSOT is especially useful in research of rTBI models for screening therapeutics, and with further technological improvements may be extended for use in rTBI patients.
CC chemokines family in fibrosis and aging: From mechanisms to therapy
2023, Ageing Research Reviews
Fibrosis is a universal aging-related pathological process in the different organ, but is actually a self-repair excessive response. To date, it still remains a large unmet therapeutic need to restore injured tissue architecture without detrimental side effects, due to the limited clinical success in the treatment of fibrotic disease. Although specific organ fibrosis and the associated triggers have distinct pathophysiological and clinical manifestations, they often share involved cascades and common traits, including inflammatory stimuli, endothelial cell injury, and macrophage recruitment. These pathological processes can be widely controlled by a kind of cytokines, namely chemokines. Chemokines act as a potent chemoattractant to regulate cell trafficking, angiogenesis, and extracellular matrix (ECM). Based on the position and number of N-terminal cysteine residues, chemokines are divided into four groups: the CXC group, the CX3C group, the (X)C group, and the CC group. The CC chemokine classes (28 members) is the most numerous and diverse subfamily of the four chemokine groups. In this Review, we summarized the latest advances in the understanding of the importance of CC chemokine in the pathogenesis of fibrosis and aging and discussed potential clinical therapeutic strategies and perspectives aimed at resolving excessive scarring formation.
Dendrimers in the context of targeting central nervous system disorders
2022, Journal of Drug Delivery Science and Technology
Citation Excerpt :
The significant role of several macrophage inflammatory protein-1 family members, such as CCL3, CCL4, CCL9, CCL20, and their receptors such as CCR1, CCR5, and CCR6 has been established before [107]. In a study, a gene silencing strategy was taken to attenuate CCL20 and CCR6 before MScs delivery in the rabbit model by delivering shRNA [108]. The researchers utilized nano dendriplexes, G4 PAMAM dendrimers complexed with plasmids to target and deliver the cargo to activated microglia.
The treatment of central nervous system disorders, such as Alzheimer's, Parkinson's, traumatic brain injury, brain cancer, etc. is challenging due to the stringent blood-brain barrier (BBB) that restricts the entry of drugs or therapeutic agents. Few invasive strategies, such as reversible disruption of BBB or direct delivery of drug-loaded nanocarriers are effective but suffer from a lack of patient compliance and other drawbacks. Among the non-invasive strategies of systemic delivery of drugs crossing the BBB, dendrimers have gained the significant interest of researchers. Dendrimers are branched three-dimensional polymeric nanostructures with a large number of functional groups that can be modified for tailored-made functionality. The versatility, biocompatibility, capacity to load drugs into the core and surface, and nanosize are the notable advantages of dendrimers. Ligand conjugated dendrimers can cross the BBB and enhance the internalization of the drugs in the target tissues of the brain. A handful of research has established the effectiveness of dendrimers, alone or drug-loaded in several CNS disorders. However, their entry into the clinical trial phase is still restricted. The original review analyzes the favorable outcome of the brain-targeted dendrimer-based research and the associated challenges to their clinical success.
Construction of Curcumin-Loaded Hydrogels for Treatment of Traumatic Brain Injury
2023, ACS Applied Polymer Materials

View all citing articles on Scopus

View full text

Published by Elsevier Inc.

Original ArticleTreatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury

Highlights

Abstract

Graphical Abstract

Section snippets

Construction of CCL20/CCR6 shRNA expression vector

Preparation and characterization of dendriplex nanoparticles

Discussion

Acknowledgment

Authors' contributions

Am J Prev Med

Alzheimers Dement

Lancet

J Control Release

Neurotherapeutics

Life Sci

J Am Coll Cardiol

Cytokine Growth Factor Rev

Int J Biochem Cell Biol

Life Sci

J Control Release

Int J Pharm

Toxicol Lett

Trends Neurosci

Front Neurol

Neurosci Biobehav Rev

Cognitive functioning ten years following traumatic brain injury and rehabilitation

Neuropsychology

Functional outcome 10 years after traumatic brain injury: its relationship with demographic, injury severity, and cognitive and emotional status

J Int Neuropsychol Soc

Traumatic brain injury in Iraq and Afghanistan veterans: new results from a national random sample study

J Neuropsychiatry Clin Neurosci

Repetitive traumatic brain injury, psychological symptoms, and suicide risk in a clinical sample of deployed military personnel

JAMA Psychiat

Long-term effects of traumatic brain injury on anxiety-like behaviors in mice: behavioral and neural correlates

Front Behav Neurosci

Mild traumatic brain injury in U.S. soldiers returning from Iraq

N Engl J Med

Traumatic brain injury screening: preliminary findings in a US army brigade combat team

J Head Trauma Rehabil

Neuroprotection in traumatic brain injury: mesenchymal stromal cells can potentially overcome some limitations of previous clinical trials

Front Neurol

J. Juranek, and S. Bedi, Clinical trials in traumatic brain injury: cellular therapy and outcome measures

Transfusion

Effect of erythropoietin and transfusion threshold on neurological recovery after traumatic brain injury: a randomized clinical trial

JAMA

A clinical trial of progesterone for severe traumatic brain injury

N Engl J Med

Mesenchymal stem cell therapy modulates the inflammatory response in experimental traumatic brain injury

Neurol Res Int

Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury

Bone Marrow Transplant

Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

Stem Cell Res Ther

A phase I clinical trial of the treatment of Crohn's fistula by adipose mesenchymal stem cell transplantation

Dis Colon Rectum

Original Article
Treatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury