Limits...
Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

Camelo S, Lajavardi L, Bochot A, Goldenberg B, Naud MC, Fattal E, Behar-Cohen F, de Kozak Y - Mol. Vis. (2007)

Bottom Line: In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected.Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment.In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

View Article: PubMed Central - PubMed

Affiliation: Center de Recherche des Cordeliers, Université Pierre et Marie Curie - Paris, Paris, France.

ABSTRACT

Purpose: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats.

Methods: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy.

Results: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP.

Conclusions: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Show MeSH

Related in: MedlinePlus

VIP-loaded rhodamine-conjugated-liposomes (VIP-Rh-lip) internalization and VIP expression by ED3-positive macrophages in cervical LN 24 h following IVT injection of VIP-Rh-Lip. A: Free VIP, detected with rabbit anti-VIP antibody (blue) localized within cells containing Rh-Lip (red; B) and expressing ED3 green (C). D: Merge image showing membranous expression of ED3 by cells containing Rh-lip and blue granules within liposomes. The bar in D represents 20 μm in all images. Confocal microscopy optical section is 2 μm in all images. Representative images of two experiments performed on cervical LN from two rats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2268712&req=5

f7: VIP-loaded rhodamine-conjugated-liposomes (VIP-Rh-lip) internalization and VIP expression by ED3-positive macrophages in cervical LN 24 h following IVT injection of VIP-Rh-Lip. A: Free VIP, detected with rabbit anti-VIP antibody (blue) localized within cells containing Rh-Lip (red; B) and expressing ED3 green (C). D: Merge image showing membranous expression of ED3 by cells containing Rh-lip and blue granules within liposomes. The bar in D represents 20 μm in all images. Confocal microscopy optical section is 2 μm in all images. Representative images of two experiments performed on cervical LN from two rats.

Mentions: VIP immunostaining was performed on frozen sections of cervical LN of normal animals that received a single IVT injection of VIP-Rh-Lip. Soluble VIP (green) was detected in the sinus of the LN (asterisks in Figure 5A,B,D). In addition, VIP was detected within large cells containing Rh-Lip corresponding to subcapsular sinus macrophages (Figure 5A-D). VIP was also detected in small, round cells (characterized in Figure 6) that contained only a minute amount of Rh-Lip (arrow in Figure 5B-D) close to VIP-Rh-Lip-bearing macrophages in the subcapsular sinus. In animals that received an IVT injection of Rh-Lip that did not contain VIP, there was no VIP detectable in the subcapsular sinus of the cervical LN (data not shown). These macrophages expressed ED3 confirming their identity as subcapsular sinus macrophages (Figure 7A-D). Our results suggest strongly that VIP travels encapsulated within liposomes from the conjunctiva (not shown) to the cervical LN (Figure 5A) where it is phagocytosed by resident macrophages.


Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

Camelo S, Lajavardi L, Bochot A, Goldenberg B, Naud MC, Fattal E, Behar-Cohen F, de Kozak Y - Mol. Vis. (2007)

VIP-loaded rhodamine-conjugated-liposomes (VIP-Rh-lip) internalization and VIP expression by ED3-positive macrophages in cervical LN 24 h following IVT injection of VIP-Rh-Lip. A: Free VIP, detected with rabbit anti-VIP antibody (blue) localized within cells containing Rh-Lip (red; B) and expressing ED3 green (C). D: Merge image showing membranous expression of ED3 by cells containing Rh-lip and blue granules within liposomes. The bar in D represents 20 μm in all images. Confocal microscopy optical section is 2 μm in all images. Representative images of two experiments performed on cervical LN from two rats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268712&req=5

f7: VIP-loaded rhodamine-conjugated-liposomes (VIP-Rh-lip) internalization and VIP expression by ED3-positive macrophages in cervical LN 24 h following IVT injection of VIP-Rh-Lip. A: Free VIP, detected with rabbit anti-VIP antibody (blue) localized within cells containing Rh-Lip (red; B) and expressing ED3 green (C). D: Merge image showing membranous expression of ED3 by cells containing Rh-lip and blue granules within liposomes. The bar in D represents 20 μm in all images. Confocal microscopy optical section is 2 μm in all images. Representative images of two experiments performed on cervical LN from two rats.
Mentions: VIP immunostaining was performed on frozen sections of cervical LN of normal animals that received a single IVT injection of VIP-Rh-Lip. Soluble VIP (green) was detected in the sinus of the LN (asterisks in Figure 5A,B,D). In addition, VIP was detected within large cells containing Rh-Lip corresponding to subcapsular sinus macrophages (Figure 5A-D). VIP was also detected in small, round cells (characterized in Figure 6) that contained only a minute amount of Rh-Lip (arrow in Figure 5B-D) close to VIP-Rh-Lip-bearing macrophages in the subcapsular sinus. In animals that received an IVT injection of Rh-Lip that did not contain VIP, there was no VIP detectable in the subcapsular sinus of the cervical LN (data not shown). These macrophages expressed ED3 confirming their identity as subcapsular sinus macrophages (Figure 7A-D). Our results suggest strongly that VIP travels encapsulated within liposomes from the conjunctiva (not shown) to the cervical LN (Figure 5A) where it is phagocytosed by resident macrophages.

Bottom Line: In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected.Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment.In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

View Article: PubMed Central - PubMed

Affiliation: Center de Recherche des Cordeliers, Université Pierre et Marie Curie - Paris, Paris, France.

ABSTRACT

Purpose: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats.

Methods: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy.

Results: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP.

Conclusions: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Show MeSH
Related in: MedlinePlus