4 research outputs found
Membrane-Induced p<i>K</i><sub>a</sub> Shifts in <i>wt</i>-pHLIP and Its L16H Variant
The pH (low) insertion peptides (pHLIPs)
is a family of peptides
that are able to insert into a lipid bilayer at acidic pH. The molecular
mechanism of pHLIPs insertion, folding, and stability in the membrane
at low pH is based on multiple protonation events, which are challenging
to study at the molecular level. More specifically, the relation between
the experimental p<i>K</i> of insertion (p<i>K</i><sup>exp</sup>) of pHLIPs and the p<i>K</i><sub>a</sub> of the key residues is yet to be clarified. We carried out a computational
study, complemented with new experimental data, and established the
influence of (de)protonation of titrable residues on the stability
of the peptide membrane-inserted state. Constant-pH molecular dynamics
simulations were employed to calculate the p<i>K</i><sub>a</sub> values of these residues along the membrane normal. In the <i>wt</i>-pHLIP, we identified Asp14 as the key residue for the
stability of the membrane-inserted state, and its p<i>K</i><sub>a</sub> value is strongly correlated with the experimental p<i>K</i><sup>exp</sup> measured in thermodynamics studies. Also,
in order to narrow down the pH range at which pHLIP is stable in the
membrane, we designed a new pHLIP variant, L16H, where Leu in the
16th position was replaced by a titrable His residue. Our results
showed that the L16H variant undergoes two transitions. The calculated
p<i>K</i><sub>a</sub> and experimentally observed p<i>K</i><sup>exp</sup> values are in good agreement. Two distinct
p<i>K</i><sup>exp</sup> values delimit a pH range where
the L16H peptide is stably inserted in the membrane, while, outside
this range, the membrane-inserted state is destabilized and the peptide
exits from the bilayer. pHLIP peptides have been successfully used
to target cancer cells for the delivery of diagnostics and therapeutic
agents to acidic tumors. The fine-tuning of the stability of the pHLIP
inserted state and its restriction to a narrow well-defined pH range
might allow the design of new peptides, able to discriminate between
tissues with different extracellular pH values
Targeting Breast Tumors with pH (Low) Insertion Peptides
Extracellular
acidity is associated with tumor progression. Elevated glycolysis
and acidosis promote the appearance of aggressive malignant cells
with enhanced multidrug resistance. Thus, targeting of tumor acidity
can open new avenues in diagnosis and treatment of aggressive tumors
and targeting metastatic cancers cells within a tumor. pH (low) insertion
peptides (pHLIPs) belong to the class of pH-sensitive agents capable
of delivering imaging and/or therapeutic agents to cancer cells within
tumors. Here, we investigated targeting of highly metastatic 4T1 mammary
tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)634Mul
transgenic mice with three fluorescently labeled pHLIP variants including
well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs.
The Var3- and Var7-pHLIPs constructs have faster blood clearance than
the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of
the above breast tumor models with tumor accumulation increasing over
4 h postinjection. Staining of nonmalignant stromal tissues in transgenic
mice was minimal. The pHLIPs distribution in tumors showed colocalization
with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest
degree of colocalization of fluorescent pHLIPs was shown to be with
lactate dehydrogenase A, which is related to lactate production and
acidification of tumors. In sum, the pHLIP-based targeting of breast
cancer presents an opportunity to monitor metabolic changes, and to
selectively deliver imaging and therapeutic agents to tumors
pH-sensitive pHLIP<sup>®</sup> coated niosomes
<p>Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80–90 nm in diameter) using nonionic surfactants Span20 (43–45 mol%), cholesterol (50 mol%) and 5 mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2–3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.</p
pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery <i>In Vivo</i>
We have developed an improved tool
for imaging acidic tumors by
reporting the insertion of a transmembrane helix: the pHLIP-<u>F</u>luorescence <u>I</u>nsertion <u>RE</u>porter (pHLIP-FIRE). In acidic tissues, such as tumors,
peptides in the pHLIP family insert as α-helices across cell
membranes. The cell-inserting end of the pHLIP-FIRE peptide has a
fluorophore–fluorophore or fluorophore–quencher pair.
A pair member is released by disulfide cleavage after insertion into
the reducing environment inside a cell, resulting in dequenching of
the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced
upon cell-insertion in the targeted tissues but is suppressed elsewhere
due to quenching. Targeting studies in mice bearing breast tumors
show strong signaling by pHLIP-FIRE, with a contrast index of ∼17,
demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo
translocation <i>in vivo</i>. Imaging and targeted cargo
delivery should each have clinical applications