Limits...
An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance.

Abed N, Saïd-Hassane F, Zouhiri F, Mougin J, Nicolas V, Desmaële D, Gref R, Couvreur P - Sci Rep (2015)

Bottom Line: The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances.Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells.Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmacy, Institut Galien UMR CNRS 8612, University of Paris-Sud XI, 5 Rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France.

ABSTRACT
The "Golden era" of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

No MeSH data available.


Related in: MedlinePlus

Intracellular survival of Staphylococcus aureus in RAW 264.7 cells after NPs treatment.(A) Infected RAW 264.7 macrophages were incubated (20 h) or not with PenG, FaPenG NPs or GePenG-SB NPs at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages. (B) Confocal microscopy of viable/dead intracellular bacteria (S. aureus) in RAW264.7 cells after 6 h treatment with PenG (B), FaPenG NPs (C) or GePenG-SB NPs (D) at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages (A). After gentamicin treatment, cells were stained with LIVE/DEAD BacLight® kit. Living intracellular bacteria are labelled in green. Dead intracellular bacteria are labelled in red. Scale bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4550931&req=5

f4: Intracellular survival of Staphylococcus aureus in RAW 264.7 cells after NPs treatment.(A) Infected RAW 264.7 macrophages were incubated (20 h) or not with PenG, FaPenG NPs or GePenG-SB NPs at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages. (B) Confocal microscopy of viable/dead intracellular bacteria (S. aureus) in RAW264.7 cells after 6 h treatment with PenG (B), FaPenG NPs (C) or GePenG-SB NPs (D) at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages (A). After gentamicin treatment, cells were stained with LIVE/DEAD BacLight® kit. Living intracellular bacteria are labelled in green. Dead intracellular bacteria are labelled in red. Scale bars = 20 μm.

Mentions: The antimicrobial activity of GePenG-SB and FaPenG NPs was tested in vitro against Staphylococcus aureus-infected macrophages and compared to free PenG. Practically, RAW 264.7 cells were first infected by S. aureus at multiplicity of infection (MOI) of 10, during 90 min. After cell washing and killing the remaining extracellular bacteria, cells were treated with 20 μg.mL−1 (equivalent PenG) of FaPenG NPs, GePenG-SB NPs or bare PenG. Figure 4A illustrates the antimicrobial activity obtained after 20 h of treatment. Neither FaPenG NPs nor free PenG displayed significant antimicrobial activity, confirming the resistance of intracellular S. aureus to Penicillin treatment. On the contrary, the treatment with GePenG-SB NPs resulted in a spectacular reduction (of more than 3 log compared to the free drug) in bacterial counts, which represented a reduction of intracellular bacteria replication of more than 99.9% compared to the untreated infected-cells. To confirm the antimicrobial activity, additional confocal microscopy analyses was performed on infected-cells treated or not with FaPenG NPs, GePenG-SB NPs or free PenG. Using the LIVE/DEAD BacLight bacterial viability assay, it was possible to distinguish between dead bacteria (labelled in red) and living ones (labelled in green). As shown on Fig. 4B, when incubated with free PenG or FaPenG NPs, most of the intracellular bacteria remained still alive, while treatment with GePenG-SB NPs, succeeded to kill most of intracellular bacteria, as soon as 6 h after treatment.


An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance.

Abed N, Saïd-Hassane F, Zouhiri F, Mougin J, Nicolas V, Desmaële D, Gref R, Couvreur P - Sci Rep (2015)

Intracellular survival of Staphylococcus aureus in RAW 264.7 cells after NPs treatment.(A) Infected RAW 264.7 macrophages were incubated (20 h) or not with PenG, FaPenG NPs or GePenG-SB NPs at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages. (B) Confocal microscopy of viable/dead intracellular bacteria (S. aureus) in RAW264.7 cells after 6 h treatment with PenG (B), FaPenG NPs (C) or GePenG-SB NPs (D) at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages (A). After gentamicin treatment, cells were stained with LIVE/DEAD BacLight® kit. Living intracellular bacteria are labelled in green. Dead intracellular bacteria are labelled in red. Scale bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4550931&req=5

f4: Intracellular survival of Staphylococcus aureus in RAW 264.7 cells after NPs treatment.(A) Infected RAW 264.7 macrophages were incubated (20 h) or not with PenG, FaPenG NPs or GePenG-SB NPs at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages. (B) Confocal microscopy of viable/dead intracellular bacteria (S. aureus) in RAW264.7 cells after 6 h treatment with PenG (B), FaPenG NPs (C) or GePenG-SB NPs (D) at 20 μg.mL−1, equiv. PenG. Control was untreated infected-macrophages (A). After gentamicin treatment, cells were stained with LIVE/DEAD BacLight® kit. Living intracellular bacteria are labelled in green. Dead intracellular bacteria are labelled in red. Scale bars = 20 μm.
Mentions: The antimicrobial activity of GePenG-SB and FaPenG NPs was tested in vitro against Staphylococcus aureus-infected macrophages and compared to free PenG. Practically, RAW 264.7 cells were first infected by S. aureus at multiplicity of infection (MOI) of 10, during 90 min. After cell washing and killing the remaining extracellular bacteria, cells were treated with 20 μg.mL−1 (equivalent PenG) of FaPenG NPs, GePenG-SB NPs or bare PenG. Figure 4A illustrates the antimicrobial activity obtained after 20 h of treatment. Neither FaPenG NPs nor free PenG displayed significant antimicrobial activity, confirming the resistance of intracellular S. aureus to Penicillin treatment. On the contrary, the treatment with GePenG-SB NPs resulted in a spectacular reduction (of more than 3 log compared to the free drug) in bacterial counts, which represented a reduction of intracellular bacteria replication of more than 99.9% compared to the untreated infected-cells. To confirm the antimicrobial activity, additional confocal microscopy analyses was performed on infected-cells treated or not with FaPenG NPs, GePenG-SB NPs or free PenG. Using the LIVE/DEAD BacLight bacterial viability assay, it was possible to distinguish between dead bacteria (labelled in red) and living ones (labelled in green). As shown on Fig. 4B, when incubated with free PenG or FaPenG NPs, most of the intracellular bacteria remained still alive, while treatment with GePenG-SB NPs, succeeded to kill most of intracellular bacteria, as soon as 6 h after treatment.

Bottom Line: The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances.Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells.Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmacy, Institut Galien UMR CNRS 8612, University of Paris-Sud XI, 5 Rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France.

ABSTRACT
The "Golden era" of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

No MeSH data available.


Related in: MedlinePlus