Control Growth Factor Release Using a Self-Assembled [polycation∶heparin] Complex

The importance of growth factors has been recognized for over five decades; however their utilization in medicine has yet to be fully realized. This is because free growth factors have short half-lives in plasma, making direct injection inefficient. Many growth factors are anchored and protected by sulfated glycosaminoglycans in the body. We set out to explore the use of heparin, a well-characterized sulfated glycosaminoglycan, for the controlled release of fibroblast growth factor-2 (FGF-2). Heparin binds a multitude of growth factors and maintains their bioactivity for an extended period of time. We used a biocompatible polycation to precipitate out the [heparin∶FGF-2] complex from neutral buffer to form a release matrix. We can control the release rate of FGF-2 from the resultant matrix by altering the molecular weight of the polycation. The FGF-2 released from the delivery complex maintained its bioactivity and initiated cellular responses that were at least as potent as fresh bolus FGF-2 and fresh heparin stabilized FGF-2. This new delivery platform is not limited to FGF-2 but applicable to the large family of heparin-binding growth factors

Release kinetics of FGF-2 in PBS and in the presence of serum proteins.

<p>[A] Release kinetics as examined by measuring the amount of ¹²⁵I-FGF-2 released from complexes. The percent of FGF-2 released from complexes was monitored over 28 days. Two different molecular weight species of PAGS were used to characterize whether release kinetics were controllable. The LMW species of PAGS released nearly 20% of its loaded growth factor, while the HMW species of PAGS released approximately 50% of incorporated growth factor over the same period of time. [B] The addition of BSA had minor impact on the release FGF-2, which is consistent with the low affinity of BSA with heparin. On the other hand, negatively charged serum proteins in FBS formed precipitate surrounding the delivery matrix, which decreased the FGF-2 release rate.</p

The bioactivity of the FGF-2 released from the [PAGS∶heparin] delviery matrix is higher than bolus FGF-2 and matches that of heparin-protected FGF-2.

<p>Note that the growth factor in the release media were 1 and 3 days old, whereas the positive controls were fresh. [A] The mitogenic potency of the released FGF-2 from days 1 and 3 examined using HUVECs indicated that the bioactivity of FGF-2 is well maintained and matched that of the fresh heparin-protected FGF-2. [B] The released FGF-2 demonstrated ability to stimulate endothelial tube formation as well as fresh heparin-protected FGF-2 and is the only group that was statistically significantly better than blous FGF-2 in HAECs. Representative phase contrast micrographs of HUVECs incubated in: [C] no FGF-2, [D] fresh bolus FGF-2, [E] fresh [heparin∶FGF-2], and [F] [PAGS∶heparin∶FGF-2] release media. Statistical significance between the negative control and other experimental groups was noted as “*”, p<0.05. <b>#</b> denotes the statistical significance between an experimental group and the bolus FGF-2 group, p<0.05.</p

The interaction between PAGS and heparin.

<p>[A] The binding of PAGS and heparin resulted in a white precipitate when combined in an aqueous solution. [B] The titration of heparin using PAGS as monitored by zeta potential measurements. The complex was nearly neutral at a 35/1 mass ratio of PAGS/heparin. [C] SEM images revealed the [PAGS∶heparin] complexes as a matrix composed of fibers and sheets, and beads (1000×). [D] Higher magnification (25000×) revealed that many of the fiber are sub-micron in diameter and the beads were in fact rings.</p

Our delivery strategy was inspired by the interaction among growth factor, heparin and growth factor receptor.

<p><i>Left</i>, the crystal structure of the [FGF∶Heparin∶FGFR] complex kindly provided by Dr. Pellegrini. The proteins are shown as coils and heparin as a stick model. The heparin-binding domains of FGFR and FGF are highlighted in pink and yellow respectively. Both analyses showed that the heparin-binding regions contain a high density of positively charged amino acid residues such as arginine. <i>Right</i>, a possible model of the matrix formed by ionic interactions between an arginine-based synthetic polycation and a [heparin∶growth factor] complex.</p

Complex loading efficiency and capacity was investigated using ¹²⁵I-FGF-2.

<p>[A] Loading efficiencies of different molecular weight PAGS was investigated for different [PAGS∶heparin] ratios. The higher molecular weight PAGS was more efficient at incorporating FGF-2 at all [PAGS∶heparin] ratios than the lower molecular weight polymer. A ratio of [35∶1] was the most efficient at incorporating FGF-2 for both molecular weight species. [B] The loading capacity of complexes was investigated for a [35∶1] ratio of low molecular weight PAGS. This ratio demonstrated a loading efficiency of 50% for all amounts of FGF-2. Statistical significance between [35∶1] and other ratios was noted as “*”, p<0.05.</p

Icam-1 targeted nanogels loaded with dexamethasone alleviate pulmonary inflammation.

Lysozyme dextran nanogels (NG) have great potential in vitro as a drug delivery platform, combining simple chemistry with rapid uptake and cargo release in target cells with "stealth" properties and low toxicity. In this work, we study for the first time the potential of targeted NG as a drug delivery platform in vivo to alleviate acute pulmonary inflammation in animal model of LPS-induced lung injury. NG are targeted to the endothelium via conjugation with an antibody (Ab) directed to Intercellular Adhesion Molecule-1(ICAM-NG), whereas IgG conjugated NG (IgG-NG) are used for control formulations. The amount of Ab conjugated to the NG and distribution in the body after intravenous (IV) injection have been quantitatively analyzed using a tracer isotope-labeled [125I]IgG. As a proof of concept, Ab-NG are loaded with dexamethasone, an anti-inflammatory therapeutic, and the drug uptake and release kinetics are measured by HPLC. In vivo studies in mice showed that: i) ICAM-NG accumulates in mouse lungs (∼120% ID/g vs ∼15% ID/g of IgG-NG); and, ii) DEX encapsulated in ICAM-NG, but not in IgG-NG practically blocks LPS-induced overexpression of pro-inflammatory cell adhesion molecules including ICAM-1 in the pulmonary inflammation

Acute and Chronic Shear Stress Differently Regulate Endothelial Internalization of Nanocarriers Targeted to Platelet-Endothelial Cell Adhesion Molecule‑1

Intracellular delivery of nanocarriers (NC) is controlled by their design and target cell phenotype, microenvironment, and functional status. Endothelial cells (EC) lining the vascular lumen represent an important target for drug delivery. Endothelium <i>in vivo</i> is constantly or intermittently (as, for example, during ischemia-reperfusion) exposed to blood flow, which influences NC–EC interactions by changing NC transport properties, and by direct mechanical effects upon EC mechanisms involved in NC binding and uptake. EC do not internalize antibodies to marker glycoprotein PECAM(CD31), yet internalize multivalent NC coated with PECAM antibodies (anti-PECAM/NC) <i>via</i> a noncanonical endocytic pathway distantly related to macropinocytosis. Here we studied the effects of flow on EC uptake of anti-PECAM/NC spheres (∼180 nm diameter). EC adaptation to chronic flow, manifested by cellular alignment with flow direction and formation of actin stress fibers, inhibited anti-PECAM/NC endocytosis consistent with lower rates of anti-PECAM/NC endocytosis <i>in vivo</i> in arterial compared to capillary vessels. Acute induction of actin stress fibers by thrombin also inhibited anti-PECAM/NC endocytosis, demonstrating that formation of actin stress fibers impedes EC endocytic machinery. In contrast, acute flow without stress fiber formation, stimulated anti-PECAM/NC endocytosis. Anti-PECAM/NC endocytosis did not correlate with the number of cell-bound particles under flow or static conditions. PECAM cytosolic tail deletion and disruption of cholesterol-rich plasmalemma domains abrogated anti-PECAM/NC endocytosis stimulation by acute flow, suggesting complex regulation of a flow-sensitive endocytic pathway in EC. The studies demonstrate the importance of the local flow microenvironment for NC uptake by the endothelium and suggest that cell culture models of nanoparticle uptake should reflect the microenvironment and phenotype of the target cells

Delivering Nanoparticles to Lungs while Avoiding Liver and Spleen through Adsorption on Red Blood Cells

Nanoparticulate drug delivery systems are one of the most widely investigated approaches for developing novel therapies for a variety of diseases. However, rapid clearance and poor targeting limit their clinical utility. Here, we describe an approach to harness the flexibility, circulation, and vascular mobility of red blood cells (RBCs) to simultaneously overcome these limitations (cellular hitchhiking). A noncovalent attachment of nanoparticles to RBCs simultaneously increases their level in blood over a 24 h period and allows transient accumulation in the lungs, while reducing their uptake by liver and spleen. RBC-adsorbed nanoparticles exhibited ∼3-fold increase in blood persistence and ∼7-fold higher accumulation in lungs. RBC-adsorbed nanoparticles improved lung/liver and lung/spleen nanoparticle accumulation by over 15-fold and 10-fold, respectively. Accumulation in lungs is attributed to mechanical transfer of particles from the RBC surface to lung endothelium. Independent tracing of both nanoparticles and RBCs <i>in vivo</i> confirmed that RBCs themselves do not accumulate in lungs. Attachment of anti-ICAM-1 antibody to the exposed surface of NPs that were attached to RBCs led to further increase in lung targeting and retention over 24 h. Cellular hitchhiking onto RBCs provides a new platform for improving the blood pharmacokinetics and vascular delivery of nanoparticles while simultaneously avoiding uptake by liver and spleen, thus opening the door for new applications

Schematic illustrating the conjugation of Ab to NG and loading of DEX.

<p>Schematic illustrating the conjugation of Ab to NG and loading of DEX.</p