Contact-facilitated drug delivery with Sn2 lipase labile prodrugs optimize targeted lipid nanoparticle drug delivery

Abstract

Sn2 lipase labile phospholipid prodrugs in conjunction with contact-facilitated drug delivery offer an important advancement in Nanomedicine. Many drugs incorporated into nanosystems, targeted or not, are substantially lost during circulation to the target. However, favorably altering the pharmacokinetics and volume of distribution of systemic drug delivery can offer greater efficacy with lower toxicity, leading to new prolonged-release nanoexcipients. However, the concept of achieving Paul Erhlich's inspired vision of a 'magic bullet' to treat disease has been largely unrealized due to unstable nanomedicines, nanosystems achieving low drug delivery to target cells, poor intracellular bioavailability of endocytosed nanoparticle payloads, and the substantial biological barriers of extravascular particle penetration into pathological sites. As shown here, Sn2 phospholipid prodrugs in conjunction with contact-facilitated drug delivery prevent premature drug diffusional loss during circulation and increase target cell bioavailability. The Sn2 phospholipid prodrug approach applies equally well for vascular constrained lipid-encapsulated particles and micelles the size of proteins that penetrate through naturally fenestrated endothelium in the bone marrow or thin-walled venules of an inflamed microcirculation. At one time Nanomedicine was considered a 'Grail Quest' by its loyal opposition and even many in the field adsorbing the pains of a long-learning curve about human biology and particles. However, Nanomedicine with innovations like Sn2 phospholipid prodrugs has finally made 'made the turn' toward meaningful translational success.

Figure 1

Contact-facilitated drug delivery illustrated with rhodamine PFOB nanoparticle bound to C32 melanoma cell (transfected with Rab GFP endocytic markers). (Reprinted with permission from Refs 26 and 27. Copyright 2002 and 2008)

Figure 2

SEM showing PFOB nanoparticle hemifusion complex to C32 melanoma cell. (Reprinted with permission from Ref . Copyright 2009)

Figure 3

(a) Three-dimensional MR angiogenesis maps of control and integrin-targeted fumagillin NP in Vx2 model. (b) Angiogenesis contrast before and 1 week after a fumagillin or control NPs in hyperlipidemic rabbits. (c) Decreased arthritic score and ankle thickness following targeted fumagillin in the K/BxN model of inflammatory arthritis. (Reprinted with permission from Refs –. Copyright 2008, 2006, and 2009)

Figure 4

Schematic representation of contact-facilitated drug delivery mechanism with Sn2 lipase labile fumagillin prodrug inserted into the lipid membrane of a PFC nanoparticle. Ligand tethering of the nanoparticle to the target cell support the formation of a hemifusion complex between the two membrane surfaces. Translation of the Fum-PD into the inner leaflet of the target cell membrane leads to its distribution through the cell except the mitochondria. Regioselective enzymatic cleavage at the Sn2 site by phospholipase (such as phospholipase A2, PLA2) liberates the drug into the cytosol. (Reprinted with permission from Ref . Copyright 2012)

Figure 5

In vivo PA images of the Matrigel™ plug area implanted in four groups of mice at 18 days using α_vβ₃-CuNPs. (a, b) Targeted CuNPs group. The enhanced neovasculature by Cu Oleate NPs are marked by arrows in (b). (c, d): Nontargeted CuNPs group. (e, f) Competition group: mice received a competitive dose α_vβ₃-oil only NP (1:1) 10 min before α_vβ₃-CuNPs. (g, h) Fum-PD group: mice received α_vβ₃-CuNPs with Fum-PD 11 and 15 days after the Matrigel™ implantation then α_vβ₃-CuNPs w/o fum-PD on day 18 for PA imaging. For all PA images, laser wavelength = 767 nm. (Reprinted with permission from Ref . Copyright 2015)

Figure 6

α_vβ₃-Fum-PD NP suppressed inflammatory arthritis in the KRN model. Targeted particle with and without drug were administered on days 2, 3, and 4 (arrows). Changes in ankle thickness (a), arthritic score (b), and body weight (c) were monitored daily. Histology on day 7 for inflammatory cell number per high power field (HPF; d, e, j), erosions (f, g, k), and proteoglycan depletion (h, i, l) ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. (Reprinted with permission from Ref . Copyright 2012)

Figure 7

α_vβ₃-Fum-PD NP (1X) in combination with MTX suppressed inflammatory arthritis in the KRN mouse model. α_vβ₃-Fum-PD NP or α_vβ₃-control NP were given on day 2 (n = 5/group) and changes in ankle thickness (a) arthritic score (b) and body weight (c) were monitored daily. Histology on day 9 revealed significant benefit for inflammatory cell number/HPF (d), erosions (e), and proteoglycan depletion (f).^*P < 0.05.

Figure 8

α_vβ₃-Paclitaxel-PD NP suppressed inflammatory arthritis in the KRN model. Targeted particle with and without drug were administered on days 2, 3, and 4 (arrows). Changes in ankle thickness (a) and arthritic score (b) were monitored daily ^** P < 0.01.

Figure 9

Mouse vascular endothelial cells (2F2B) stimulated with angiotensin II (1nM) were treated with paclitaxel, docetaxel, paclitaxel prodrug, or docetaxel prodrug at 0.5, 1, 5, 10, 50, and 100 µg/mL for 1 h. Cultures were monitored for proliferation at 24, 48, and 72 h. No difference (P = NS) in biopotency of the different taxanes forms was detected. (Reprinted with permission from Ref . Copyright 2014)

Figure 10

(a) T1w MR images of Vx 2 tumor obtained at baseline and 3 hours after α_vβ₃-Gd-DOTA NP in animals receiving α_vβ₃-no drug NP showing peripheral tumor angiogenesis contrast enhancement false colored in yellow. (b) The same image sequence as panel (a) in Vx2 tumor rabbits following α_vβ₃-Dxtl-PD NP. (c) Histogram illustrating marked angiogenesis in the control rabbits rim (tumor peripheral 50%) versus those receiving Dtxl-PD with or without targeting. Note minimal contrast signal with the tumor core. Marked decrease in contrast in nontargeted (NT) animal group suggests that the dosage or frequency of treatment was in excess of the therapeutic need with passive entrapment providing adequate particle-to-endothelium contact to afford significant antiangiogenesis. (d) Representative three-dimensional neovascular maps of Vx2 tumor in control and targeted Dxtl-PD treatment groups. Note asymmetric distribution of confluent neovascular regions in the control rabbit note appreciated in the treated animal. Blue voxels equate to α_vβ₃-Gd-DOTA NP contrast enhancement exceeding three standard deviations over baseline. ^*P < 0.05, ^**P < 0.01. (Reprinted with permission from Ref . Copyright 2014)

Figure 11

(a) Histogram showing a trend (nonsignificant [NS], P = 0.2) toward increased apoptosis among Vx2 tumor rabbits receiving α_vβ₃-Dxtl-PD NP versus controls. (b) Illustrative sections (40×) of Vx2 tumor following TUNEL staining (brown arrows) using a methyl green nuclear positive reference for tumor area (green arrows). (c) Histogram showing a significant (P < 0.05) decrease in proliferation index among Vx2 tumor rabbits receiving α_vβ₃-Dxtl-PD NP versus controls. (d) Representative sections (40×) of Vx2 tumor following PCNA staining (brown arrows) using a methyl green nuclear positive reference for tumor area (green arrows). ^*P < 0.05. (Reprinted with permission from Rf . Copyright 2014)

Figure 12

(a) Chemical structures of VLA-4-PEG-phospholipid conjugate, α_vβ₃-PEG-phospholipid conjugate and MI1-PD. (b) Schematic representation illustrating nanoparticle (NP) design and lipid monolayer-mediated intracellular release of MYC-inhibitor from MI1-PD. The Myc prodrug (MI1-PD) from Panel a is represented by circles. The integrin-targeting ligands (VLA-4-PEG-phospholipid conjugate and α_vβ₃-PEG-phospholipid conjugate) from Panel a is represented by triangles. (A) Anti-Myc NPs are targeted by the homing ligand to myeloma (MM) cells expressing integrin proteins on their cell surface (represented by V). (B) Proximity induces hemifusion of lipid layers between NP and MM cells. (C) The MI1-PD in the NP membrane is then exposed to cellular lipases that (D) cleave the phospholipid moiety of the prodrug and release the anti-Myc moiety into the MM cell.

Figure 13

Myc inhibitor 1 (MI1) and MI1 prodrug (MI1-PD) decrease viability and induce apoptosis in myeloma cell lines. (a) MTT assay for cell viability at 24 h with MI1 and MI1-PD at given concentrations in H929, U266, and 5TGM1 cells. Values are average of three separate experiments in triplicates and expressed as mean ± SD. ^*P < 0.05. (b) Representative of Annexin V-PE staining of cells for apoptosis following MI1 and MI1-PD treatment (left and right columns respectively) at given concentrations in H929, U266, and 5TGM1 cells at 24 h. (Reprinted with permission from Ref . Copyright 2014)

Figure 14

Anti-MYC NPs prolong survival in a mouse model of multiple myeloma. (a) MTT studies in 5TGM1 cells at 24 h. (b) The experimental protocol for the in vivo tumor growth assay. The groups were as follows: (1) ND/NT 200; (2) T/ND 200; (3) T/D 200; (4) NT/ND 20; (5) T/ND 20; and (6) T/D 20 all injected on days 3, 5, 7, 10, 12, and 14 following the i.v. injections of 5TGM1 cells (day 0) (c, d) SPEP quantification of each treatment groups 3 days after the last injection was given (each dot represents SPEP value for one mouse). (e, f) Kaplan–Meier survival curves of the treated mice with 20 and 200 nm NPs. (Reprinted with permission from Ref . Copyright 2014)